Target capture system

ABSTRACT

The invention generally relates to a system for isolating or separating a target from a sample. In certain aspects, processes performed by the target capture system include introducing a plurality of magnetic particles, in which a plurality of the particles include at least one binding moiety specific to a target, into a sample to form at least one target/particle complex and applying a magnetic field to isolate the magnetic particle/target complexes from the sample. The process starts at inputting a sample into the system and ends at delivering a capture target or nucleic acids of the target into a container for further analysis.

RELATED APPLICATION

The present application claims the benefit of and priority to U.S.provisional patent application Ser. No. 61/739,644, filed Dec. 19, 2012,the content of which is incorporated by reference herein in itsentirety.

TECHNICAL FIELD

This invention generally relates to cartridges for separating targetsfrom a sample and methods related thereto.

BACKGROUND

Many laboratory and clinical procedures involve processing a sample toseparate a target from the sample for subsequent identification andanalysis of the target. Such processes are commonly used to detect awide range of targets, including biological entities such as cells,viruses, proteins, bacterium, nucleic acids, etc., and have applicationsin clinical diagnostics, biohazard screenings, and forensic analyses.

Often there is an immediate need to identify the target, whether todetermine the proper course of treatment or to develop response protocolfor biohazard threat. For example, blood-borne pathogens are asignificant healthcare problem because a delayed or improper diagnosiscan lead to sepsis. Sepsis, a severe inflammatory response to infection,is a leading cause of death in the United States and early detection ofthe blood-borne pathogens underlying the infection is crucial topreventing the onset of sepsis. With early detection, the pathogen'sdrug/antibiotic resistant profile can be obtained which allows theclinician to determine the appropriate anti-microbial therapy for aquicker and more effective treatment.

Pathogens during active blood-borne infection or after antibiotictreatment are typically present in minute levels per mL of body fluid.Several techniques have been developed for isolation of pathogens in abody fluid sample, which include molecular detection methods, antigendetection methods, and metabolite detection methods. These conventionalmethods often require culturing specimens, such as performing anincubation or enrichment step, in order to detect the low levels ofpathogens. The incubation/enrichment period is intended to allow for thegrowth of bacteria and an increase in bacterial cell numbers to morereadily aid in isolation and identification. In many cases, a series oftwo or three separate incubations is needed to isolate the targetbacteria. Moreover, enrichment steps require a significant amount oftime and can potentially compromise test sensitivity by killing some ofthe cells sought to be measured. In certain cases, a full week may benecessary to reach the desired levels of bacteria.

The above techniques can be carried out on microfluidic devices toseparate the pathogen or target from the sample. However, currentmicrofluidic devices require extensive sample preparation prior tointroducing the sample into the device for subsequent isolation.Microfluidic devices by definition are designed to conduct reactions andprocess at a microfluidic scale. To isolate pathogens in a microfluidicdevice, it is necessary to incubate/enrich the sample to expand thelevels of pathogens to increase the chance that the small volume ofsample introduced into the microfluidic device contains a pathogen.Alternatively, a large volume of fluid suspected of containing apathogen is introduced at an extremely slow pace through a tube or bypiecemeal pipetting the sample into the microfluidic device. Thesemethods are equally time consuming as incubation and can result in lossof clinically relevant sample (i.e. sample with sufficient levels oftargets for capture). In addition, the transfer of large volume of fluidinto the microfluidic device may expose the sample to contamination.

Currently, there is no effective device capable of rapidly andeffectively isolating a target that is fast and sensitive in order toprovide data critical for patient treatment and biohazard analysis in aclinically relevant time frame.

SUMMARY

The invention provides a system (target capture system) that allowsrapid isolation of a target analyte from a sample without the need forsample preparation or extensive manual operation. A target capturesystem of the invention concentrates essentially all of a clinicallyrelevant portion of a sample (less than 1 mL) from an initial samplevolume of about 10 mL. The ability to isolate small, clinicallyrelevant, volumes of sample improves the efficiency in which nucleicacids can be extracted from a target for subsequent analysis.

The target capture systems generally include a cartridge and aninstrument. The cartridge includes components such as channels, mixingchambers, and traps to process the sample for target isolation. Thecartridge may interface with an instrument having one or moreassemblies, such as mechanical, magnetic, pneumatic, and fluidicassemblies, that interact with the cartridge to assist/drive theprocesses performed on the cartridge. The target capture systems of theinvention are integrated to perform several processes on a sampleinputted into the cartridge to achieve a final result without usermanipulation. The final result can be capture of live/whole target cellsor isolation of nucleic acids from target cells within the sample.

In certain aspects, the processes performed by the target capture systeminclude introducing a plurality of magnetic particles, in which aplurality of the particles include at least one captured moiety specificto a target, into a sample to form a mixture that includes sample andparticles. The mixture is incubated to form at least one target/particlecomplex and a magnetic field is applied to isolate the magneticparticle/target complexes from the sample. The process starts atinputting a sample and ends at delivering a capture target (or nucleicacids of the target) into a container for further analysis.

Cartridges of the invention can include a chamber for holding andreleasing the magnetic particles into a sample to form a mixture and atleast one magnetic trap for receiving the sample/magnetic particlemixture. The magnetic trap engages with a magnetic assembly to isolatethe magnetic particles from the sample. In certain embodiments, thecartridge includes a first magnetic trap in communication with a secondmagnetic trap, in which both magnetic traps engage with a magneticassembly to isolate magnetic particles from a sample. In one embodiment,the first magnetic trap isolates magnetic particles from the sample andthe isolated magnetic particles are then transferred through the secondmagnetic trap. The second magnetic trap engages with a second magneticassembly to isolate the plurality of magnetic particles from the firstmagnetic trap. A lysing mechanism operably associated with the secondmagnetic trap can be used to lyse at least one target cell bound to atleast one of the magnetic particles within the second magnetic trap. Thecartridge may further include a matrix for receiving lysate from thefirst magnetic trap and retaining nucleic acid from the lysate. In oneembodiment, the matrix is an affinity column. The cartridge can furtherinclude a reaction chamber, such as a bubble mixer, in communicationwith the matrix for pre-treating the lysate.

In one embodiment, the cartridge is for use with an instrument and theinstrument includes a first magnetic assembly, a second magneticassembly, and a lysing mechanism. The first and second magneticassemblies generate magnetic fields to isolate magnetic particlesdisposed within a sample against a surface of a corresponding magnetictrap. The lysing mechanism may perform cell lysis on targets bound tothe magnetic particles. The lysing mechanism can be a sonication device.Lysis can be achieved by placing the sonication against a surface of amagnetic trap and generating sonic wave to invoke cell lysis on anytargets bound to the magnetic particles within the magnetic trap. Theinstrument can also include a drive mechanism for driving andcontrolling movement of the sample, the magnetic particles, and anyother fluids or substances into, within, and out of the cartridge. Thedrive mechanism can be pneumatic, mechanical, magnetic and/or fluidic.

For isolation and detection assays conducted on cartridges or chips, itis important to transfer the entire obtained sample from a collectiondevice into the cartridge to increase the efficiency of isolation ordetection. Especially in situations where there is little sample, whichis often the case in forensic analysis, or where there is a smallconcentration of targets per mL of sample (e.g. 1 CFU/mL), which isoften the case for pathogenic detection.

Accordingly, the cartridges of the invention may include acartridge/vessel interface designed to maximize the amount of sampletransferred from the vessel and into the cartridge to avoid loss ofclinically relevant sample within a sample collection vessel and/orduring sample transfer. This ensures that substantially all clinicallyrelevant sample is transferred into the cartridge and processed. Thecartridge/vessel interface places the vessel containing the sample intwo-way communication with the cartridge. The communication between thevessel and the cartridge can be pneumatic, fluidic, or both. In certainembodiments, the cartridge/vessel interface can include an input memberand an output member. The output member introduces fluids, gases, andsubstances from the cartridge into the vessel and the input membertransfers the sample and any introduced fluids, gases, and substancesfrom vessel into the cartridge. Typically, the input and output membersdefine a lumen and include a penetrating tip. The input and outputmembers may be designed to penetrate and extend into the vessel.

In certain embodiments, the cartridge includes a cartridge/vesselinterface, a chamber, and a magnetic trap. The cartridge/vesselinterface couples a vessel containing a sample to the cartridge. Thechamber is in communication with the cartridge/interface and canreleasably hold fluid or substance for introducing the fluid orsubstance into the vessel. The magnetic trap is in communication withthe vessel at the interface and receives the contents of the vessel. Thevessel contents can include the sample and the fluid or substancesintroduced into the vessel from the chamber. In one embodiment, thechamber contains a plurality of magnetic particles to release into thevessel. The magnetic particles can include one or more binding moietiesspecific to a target within the sample. The sample and magneticparticles are transferred into the magnetic trap of the cartridge. Themagnetic trap can engage with a magnetic assembly to isolate themagnetic particles from the sample.

A significant advantage of certain embodiments is that the cartridgeincludes both macrofluidic and microfluidic components so that thecartridge can process both macrofluidic and microfluidic volumes offluid. This aspect of the invention accounts for the fact that a minuteamount of target cells may be present in a sample having a macrofluidicvolume which necessitates processing the entire macrofluidic volume inorder to increase the likelihood that the target cell will be isolated.To isolate pathogens in a microfluidic device, the entire macrofluidicvolume of sample would have to be transferred slowly or in a piecemealfashion (e.g. via pipetting) into a microfluidic device at microfluidicrate, which undesirably takes a long amount of time and risks losing thetarget analyte of interest during the transfer. In certain aspects, thecartridge is designed to consolidate a sample of macrofluidic volumeinto a concentrated microfluidic volume of fluid that contains targetcells of interest. The concentrated microfluidic volume is thenprocessed at the microfluidic level.

In certain aspects, the target capture system includes a macrofluidicportion and a microfluidic portion. The macrofluidic portion includes afirst magnetic trap and is configured to process a macro-scale volume offluid including a sample, in which processing includes introducing aquantity of magnetic particles into the macro-scale volume of fluid. Themicrofluidic portion is in communication with the macrofluidic portionand includes a second magnetic trap. The microfluidic portion isconfigured to isolate a micro-scale volume of the macro-scale volume offluid in which the micro-scale volume of fluid contains substantiallythe entire quantity of magnetic particles. In certain embodiments, themagnetic particles include a binding moiety specific to a target withinthe sample. Within the cartridge, the magnetic particles initially bindto targets in a macrofluidic volume of fluid and then the magneticparticles are concentrated into a micro-scale volume of fluid. Thisallows one to isolate targets initially present in a macro-scale volumeinto an easy to process micro-scale volume of fluid.

Magnetic particles suitable for use in the target capture system areconjugated to a capture moiety specific to a target analyte. In certainembodiments, the target capture system utilizes compositions thatinclude a plurality of sets of magnetic particles conjugated to capturemoieties to capture different targets of interest. For example, each setof the plurality of magnetic particles may be conjugated with capturemoieties having different specificities for different pathogens. Thecapture moieties conjugated to the magnetic particles may be of the sameclass or different class. For example, one set of capture moieties maybe antibodies specific to a first pathogen, and the other set of capturemoieties may be other antibodies specific to a second pathogen. Inanother example, one set of capture moieties may be antibodies specificto a first pathogen, and the other set of capture moieties may belectins specific to a second pathogen. In certain embodiments, eachmagnetic particle of a set is conjugated to at least two different setsof capture moieties specific to different pathogens. That is, onemagnetic particle may have two or more capture moieties specific todifferent pathogens. The two or more capture moieties conjugated to amagnetic particle may be of the same class or different class.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 outlines the processing steps of the target capture systemaccording to certain embodiments.

FIG. 2 depicts a schematic overview of a cartridge according to certainembodiments.

FIG. 3 depicts an external view of the cartridge according to certainembodiments.

FIG. 4 depicts the cartridge/vessel interface according to certainembodiments.

FIG. 5 depicts the instrument of the target capture system according tocertain embodiments.

FIG. 6 depicts the instrument of the target capture system according tocertain embodiments.

FIG. 7 depicts a schematic overview of a cartridge according to certainembodiments.

FIGS. 8-22 depict the processing steps for target capture within thecartridge of the target capture system according to certain embodiments.

DETAILED DESCRIPTION

The invention generally relates to target capture systems configured tocarry out the processes necessary to isolate a target analyte from asample without the need for sample preparation or manual operation. Thetarget capture systems generally include a cartridge and an instrument.The cartridge includes components such as channels, reaction chamber,and reservoirs, etc. configured to perform processes for isolating atarget from a sample. The cartridge interfaces with an instrument havingone or more assemblies or subsystems, such as mechanical, magnetic,pneumatic, and fluidic assemblies, that interact with the cartridge toassist/drive the processes performed on the cartridge. The systems ofthe invention are fully integrated to perform several processes on asample inputted into the cartridge to achieve a final result, such aslive cell capture or isolated nucleic acids from a target cell, withoutuser manipulation.

Various embodiments of the target capture system including the cartridgeand the instrument and processes performed by the target capture systemare described in detail below.

In certain aspects, the processes performed by the target capturesystems generally include introducing a plurality of magnetic particles,in which each particle includes at least one binding moiety specific toa target, into a sample to form at least one target/particle complex andapplying a magnetic field to isolate the magnetic particle/targetcomplexes from the sample. The process starts at inputting a sample andends at delivering a capture target (or nucleic acids of the target)into a container for further analysis.

FIG. 1 outlines the processing steps for isolating a target using thetarget capture system according to one embodiment. In step 1100, avessel is coupled to an interface on a cartridge to place the cartridgein direct communication with the vessel. The cartridge is then loadedinto the instrument. The instrument is then activated to perform thefollowing processes. In step 1110, magnetic capture particles from thecartridge are introduced into the vessel from the cartridge through theinterface. The capture particles may include binding moieties specificto a target so that targets within the sample will bind to theparticles. In certain aspects, the capture particles are disposed withina fluid, such as a buffer, to facilitate introduction of the captureparticles and fluid in the vessel. The sample/capture particles/fluidmixture is transferred from the vessel into the cartridge. The fluidintroduced into the vessel rinses any remaining sample from the vessel.In addition, air can be introduced into the vessel to force transfer ofthe vessel contents (sample/fluid/particles) into the cartridge. In step1120, the sample/particle/fluid mixture is incubated and agitated withinthe cartridge to form target/magnetic field complexes. In step 1130, amagnetic field is applied to capture the target/magnetic field isapplied to capture the target/magnetic particles on surface of achamber/trap of the cartridge. As in steps 1140 and 1160, the surface ofthe magnetic trap is then washed with a wash solution that removes anyunbound sample and/or removes the bound targets from the magneticparticles. In step 1150, the target capture system elutes thetarget/magnetic particles complexes or targets directly into a vial forsubsequent analysis. This allows one to capture, for example, the wholecell/live cell for subsequent analysis. Alternatively and in step 1160,the target capture system can continue to process the capture target toextract and isolate nucleic acids from the target. In this embodiment,the magnetic particle/target complexes are subject to a celllysis/nucleic acid extraction step after the wash to obtain nucleicacids from the target cell. In step 1170, the resulting lysate is driventhrough an affinity column for capturing nucleic acids from the targetcells. In step 1180, the affinity column is then eluted to drivepurified nucleic acids into a vial. The vial with the isolated targetscan then be removed from the target capture system by an operator forfurther analysis as in step 1190.

In addition to the methods of target capture described herein, thetarget capture system may be utilized to isolate pathogens using othermethods, including the methods described in co-owned U.S. publicationnos. 2011/0263833, 2011/0262925, 2011/0262932, 2011/0262933,2011/0262926, and 2011/0262927, the entireties of which are incorporatedby reference.

Sample

In certain aspects, the target capture system is designed to isolatetargets from biological samples including, for example, blood, serum,plasma, buffy coat, saliva, wound exudates, pus, lung and otherrespiratory aspirates, nasal aspirates and washes, sinus drainage,bronchial lavage fluids, sputum, medial and inner ear aspirates, cystaspirates, cerebral spinal fluid, stool, diarrheal fluid, urine, tears,mammary secretions, ovarian contents, ascites fluid, mucous, gastricfluid, gastrointestinal contents, urethral discharge, synovial fluid,peritoneal fluid, meconium, vaginal fluid or discharge, amniotic fluid,penile discharge, or the like may be tested. In addition, fluidicsamples formed from swabs or lavages representative of mucosalsecretions and epithelia are acceptable, for example mucosal swabs ofthe throat, tonsils, gingival, nasal passages, vagina, urethra, rectum,lower colon, and eyes, as are homogenates, lysates and digests of tissuespecimens of all sorts. In addition to biological samples, samples ofwater, industrial discharges, food products, milk, air filtrates, and soforth are suitable for use with the target capture system. These includefood, environmental and industrial samples. In certain embodiments,fluidization of a generally solid sample may be required and is aprocess that can readily be accomplished off-cartridge.

In certain aspects, the target capture system can process macro-scaleand micro-scale volumes of fluid. Macro-scale volumes are consideredvolumes 1 mL and above and micro-scale volumes are considered volumesbelow 1 mL. The cartridge of the target capture system may be designedto directly couple to a vessel containing the sample. Vessels suitablefor use with the target capture system can be macrofluidic vessels ormicrofluidic vessels. For example, the target capture system can processa sample from a vessel that contains fluid having a volume of 1 mL to100 mL, preferably around 5 mL to 20 mL. Alternatively, the targetcapture system can process a sample from a vessel that contains a fluidhaving a volume of 10 to 500 μL. In one aspect, the cartridge of thetarget capture system is designed to couple to a 10 mL collection tube,such as a blood collection tube (e.g., VACUTAINER (test tubespecifically designed for venipuncture, commercially available fromBecton, Dickinson and company). However, the invention is not limited tocoupling with a VACUTAINER (test tube specifically designed forvenipuncture, commercially available from Becton, Dickinson and company)and can coupled with any enclosed vessel with a top that is configuredto directly couple to an interface on the cartridge. The vessel andvessel/cartridge interface are described in more detail hereinafter.

Targets

The target capture system of the invention can be used to isolate anytarget from the sample. The target refers to the substance in the samplethat will be captured and isolated by the target capture system. Thetarget may be bacteria, fungi, a protein, a cell (such as a cancer cell,a white blood cell a virally infected cell, or a fetal cell circulatingin maternal circulation), a virus, a nucleic acid (e.g., DNA or RNA), areceptor, a ligand, a hormone, a drug, a chemical substance, or anymolecule known in the art.

In certain embodiments, the target is a pathogenic bacterium, fungus orboth. Exemplary fungal species that may be captured by methods of theinvention include species from the Candida genus, Aspergillus genus, andCryptococcus genus. In particular embodiments, the specific fungalspecies include C. albicans, C. glabarata, C. parasilosis, C.tropicalis, C. krusei, Cryptococcus neoformans, Cryptococcus gattii.Exemplary bacterial species that may be captured and isolated by methodsof the invention include species from the following genera Escherichia,Listeria, Clostridium, Enterobacteriaceae, Mycobacterium, Shigella,Borrelia, Campylobacter, Bacillus, Salmonella, Enterococcus,Pneumococcus, Streptococcus, Staphylococcus, Acinetobacter,Strenotrophomonas, Pseudomanos, Neisseria, Haemophilus, Clostridium, andEnterococcus. The method may also be used to detect the mecA gene, whichis a bacterial gene associated with antibiotic resistance. In addition,the specific species of pathogen selected for capture and isolation maybe based on a certain phenotypic characteristic. Devices and methods ofthe invention may be used to isolate only gram positive bacteria from asample, or, alternatively, used to isolate only gram negative bacteriafrom a sample. In certain embodiments, devices and methods of theinvention are used to isolate both gram positive and gram negativebacteria from a sample.

Magnetic Particles

In certain aspects, the target capture system may use magnetic particlesto isolate a target from the sample. Any type of magnetic particles canbe used in conjunction with the target capture system. Production ofmagnetic particles and particles for use with the invention are known inthe art. See for example Giaever (U.S. Pat. No. 3,970,518), Senyi et al.(U.S. Pat. No. 4,230,685), Dodin et al. (U.S. Pat. No. 4,677,055),Whitehead et al. (U.S. Pat. No. 4,695,393), Benjamin et al. (U.S. Pat.No. 5,695,946), Giaever (U.S. Pat. No. 4,018,886), Rembaum (U.S. Pat.No. 4,267,234), Molday (U.S. Pat. No. 4,452,773), Whitehead et al. (U.S.Pat. No. 4,554,088), Forrest (U.S. Pat. No. 4,659,678), Liberti et al.(U.S. Pat. No. 5,186,827), Own et al. (U.S. Pat. No. 4,795,698), andLiberti et al. (WO 91/02811), the content of each of which isincorporated by reference herein in its entirety.

Magnetic particles generally fall into two broad categories. The firstcategory includes particles that are permanently magnetizable, orferromagnetic; and the second category includes particles thatdemonstrate bulk magnetic behavior only when subjected to a magneticfield. The latter are referred to as magnetically responsive particles.Materials displaying magnetically responsive behavior are sometimesdescribed as superparamagnetic. However, materials exhibiting bulkferromagnetic properties, e.g., magnetic iron oxide, may becharacterized as superparamagnetic when provided in crystals of about 30nm or less in diameter. Larger crystals of ferromagnetic materials, bycontrast, retain permanent magnet characteristics after exposure to amagnetic field and tend to aggregate thereafter due to strongparticle-particle interaction. In certain embodiments, the particles aresuperparamagnetic particles. In certain embodiments, the magneticparticle is an iron containing magnetic particle. In other embodiments,the magnetic particle includes iron oxide or iron platinum.

In certain embodiments, the magnetic particles include at least about10% superparamagnetic particles by weight, at least about 20%superparamagnetic particles by weight, at least about 30%superparamagnetic particles by weight, at least about 40%superparamagnetic particles by weight, at least about 50%superparamagnetic particles by weight, at least about 60%superparamagnetic particles by weight, at least about 70%superparamagnetic particles by weight, at least about 80%superparamagnetic particles by weight, at least about 90%superparamagnetic particles by weight, at least about 95%superparamagnetic particles by weight, or at least about 99%superparamagnetic particles by weight. In a particular embodiment, themagnetic particles include at least about 70% superparamagneticparticles by weight.

In certain embodiments, the superparamagnetic particles are less than100 nm in diameter. In other embodiments, the superparamagneticparticles are about 150 nm in diameter, are about 200 nm in diameter,are about 250 nm in diameter, are about 300 nm in diameter, are about350 nm in diameter, are about 400 nm in diameter, are about 500 nm indiameter, or are about 1000 nm in diameter. In a particular embodiment,the superparamagnetic particles are from about 100 nm to about 250 nm indiameter.

In certain embodiments, the particles are particles (e.g.,nanoparticles) that incorporate magnetic materials, or magneticmaterials that have been functionalized, or other configurations as areknown in the art. In certain embodiments, nanoparticles may be used thatinclude a polymer material that incorporates magnetic material(s), suchas nanometal material(s). When those nanometal material(s) orcrystal(s), such as Fe₃O₄, are superparamagnetic, they may provideadvantageous properties, such as being capable of being magnetized by anexternal magnetic field, and demagnetized when the external magneticfield has been removed. This may be advantageous for facilitating sampletransport into and away from an area where the sample is being processedwithout undue particle aggregation.

One or more or many different nanometal(s) may be employed, such asFe₃O₄, FePt, or Fe, in a core-shell configuration to provide stability,and/or various others as may be known in the art. In many applications,it may be advantageous to have a nanometal having as high a saturatedmoment per volume as possible, as this may maximize gradient relatedforces, and/or may enhance a signal associated with the presence of theparticles. It may also be advantageous to have the volumetric loading ina particle be as high as possible, for the same or similar reason(s). Inorder to maximize the moment provided by a magnetizable nanometal, acertain saturation field may be provided. For example, for Fe₃O₄superparamagnetic particles, this field may be on the order of about 0.3T.

The size of the nanometal containing particle may be optimized for aparticular application, for example, maximizing moment loaded upon atarget, maximizing the number of particles on a target with anacceptable detectability, maximizing desired force-induced motion,and/or maximizing the difference in attached moment between the labeledtarget and non-specifically bound targets or particle aggregates orindividual particles. While maximizing is referenced by example above,other optimizations or alterations are contemplated, such as minimizingor otherwise desirably affecting conditions.

In an exemplary embodiment, a polymer particle containing 80 wt % Fe₃O₄superparamagnetic particles, or for example, 90 wt % or highersuperparamagnetic particles, is produced by encapsulatingsuperparamagnetic particles with a polymer coating to produce a particlehaving a diameter of about 250 nm.

Binding Moiety

Magnetic particles for use with the target capture system can have atarget-specific binding moiety (capture moiety) that allows for theparticles to specifically bind the target of interest in the sample. Thecapture moiety may be any molecule known in the art and will depend onthe target to be captured and isolated. In certain embodiments, thetarget capture system utilizes compositions that include a plurality ofsets of magnetic particles conjugated to capture moieties specific todifferent targets of interest. Compositions of magnetic particles forisolating pathogens in heterogeneous samples are described in moredetail in co-owned U.S. publication no. 2011/0263833 and 2011/0262925,as well co-owned U.S. provisional app. No. 61/739,616, filed Dec. 19,2012.

The one or more different sets of magnetic particles may be coupled toone or more classes of capture moieties. Exemplary classes of capturemoieties include oligonucleotides (including nucleic acid probes),proteins, ligands, lectins, antibodies, aptamers, bactertiophages, hostinnate immunity biomarkers (e.g., CD14), host defense peptides (e.g.,defensins), bacteriocins (e.g., pyocins), and receptors. The capturemoiety may be specific to a certain species within a genus of pathogen,or the capture moiety may be generally specific to several specieswithin or the entire genus of pathogen. A class of capture moieties mayinclude one or more different types of that class, e.g. an antibodyspecific to one pathogen and an antibody specific to another pathogen.In addition, one set of magnetic particles may be conjugated to a classof capture moieties that are different from a class of capture moietiesconjugated to another set. For example, one set of magnetic particlesmay be conjugated to an antibody specific to a pathogen and another setof magnetic particles may be conjugated to a lectin specific to adifferent pathogen. The classes and types of capture moieties utilizedwill depend on the target to be captured and isolated.

In certain embodiments, each magnetic particle of a set is conjugated toat least two different sets of capture moieties specific to differentpathogens. That is, one magnetic particle may have two or more capturemoieties specific to different pathogens. The two or more capturemoieties conjugated to a magnetic particle may be of the same class ordifferent class. For example, a magnetic particle may have two or moreantibodies specific to different pathogens. In another example, amagnetic particle may be conjugated to an antibody specific to apathogen and a lectin specific to a different pathogen.

In particular embodiments, the capture moiety is an antibody, such as anantibody that binds a particular pathogen. General methodologies forantibody production, including criteria to be considered when choosingan animal for the production of antisera, are described in Harlow et al.(Antibodies, Cold Spring Harbor Laboratory, pp. 93-117, 1988). Forexample, an animal of suitable size such as goats, dogs, sheep, mice, orcamels are immunized by administration of an amount of immunogen, suchthe target bacteria, effective to produce an immune response. Anexemplary protocol is as follows. The animal is injected with 100milligrams of antigen resuspended in adjuvant, for example Freund'scomplete adjuvant, dependent on the size of the animal, followed threeweeks later with a subcutaneous injection of 100 micrograms to 100milligrams of immunogen with adjuvant dependent on the size of theanimal, for example Freund's incomplete adjuvant. Additionalsubcutaneous or intraperitoneal injections every two weeks withadjuvant, for example Freund's incomplete adjuvant, are administereduntil a suitable titer of antibody in the animal's blood is achieved.Exemplary titers include a titer of at least about 1:5000 or a titer of1:100,000 or more, i.e., the dilution having a detectable activity. Theantibodies are purified, for example, by affinity purification oncolumns containing protein G resin or target-specific affinity resin.

Polyclonal antibodies, monoclonal antibodies, or both can be conjugatedto magnetic particles in accordance with compositions and methods of theinvention. Polyclonal antibodies are antibodies that are secreted bydifferent B cell lineages, and are a collection of immunoglobulinmolecules that react against a specific antigen, each identifying adifferent eptitope. Thus, polyclonal antibodies recognize multipleepitopes on any one antigen. Polyclonal antibodies are useful inidentifying homologous pathogens, and allow for general isolation andcapture of a range of species. In contrast, monoclonal antibodies areconstructive from one cell line, and recognize only one epitope on anantigen. Monoclonal antibodies are more specific, and typically onlybind to the epitope of the specific target cell (e.g. less likely tobind to a range of species).

The technique of in vitro immunization of human lymphocytes is used togenerate monoclonal antibodies. Techniques for in vitro immunization ofhuman lymphocytes are well known to those skilled in the art. See, e.g.,Inai, et al., Histochemistry, 99(5):335 362, May 1993; Mulder, et al.,Hum. Immunol., 36(3):186 192, 1993; Harada, et al., J. Oral Pathol.Med., 22(4):145 152, 1993; Stauber, et al., J. Immunol. Methods,161(2):157 168, 1993; and Venkateswaran, et al., Hybridoma, 11(6) 729739, 1992. These techniques can be used to produce antigen-reactivemonoclonal antibodies, including antigen-specific IgG, and IgMmonoclonal antibodies.

Any antibody or fragment thereof having affinity and specific for thebacteria of interest is within the scope of the invention providedherein. Immunomagnetic particles against Salmonella are provided inVermunt et al. (J. Appl. Bact. 72:112, 1992). Immunomagnetic particlesagainst Staphylococcus aureus are provided in Johne et al. (J. Clin.Microbiol. 27:1631, 1989). Immunomagnetic particles against Listeria areprovided in Skjerve et al. (Appl. Env. Microbiol. 56:3478, 1990).Immunomagnetic particles against Escherichia coli are provided in Lundet al. (J. Clin. Microbiol. 29:2259, 1991).

In certain embodiments, the target-specific binding moiety is a lectin.Lectins are sugar-binding proteins that are highly specific for theirsugar moieties. Exemplary lectins that may be used as target-specificbinding moieties include Concanavalin (ConA), Wheat Germ Extract WGA).Lectins that specifically bind to bacteria and fungi are known, see,e.g. Stoddart, R. W., and B. M. Herbertson. “The use offluorescein-labelled lectins in the detection and identification offungi pathogenic for man: a preliminary study.” Journal of medicalmicrobiology 11.3 (1978): 315-324; and U.S. Pat. No. 5,004,699. Inaddition, other lectins that have pathogen-binding properties are shownin Table 1 below.

TABLE 1 Lectins with Carbohydrate Specificity Lectin Source CarbohydrateSpecificity AMA Arum maculatum (AMA) from ‘lords and Mannose ladies’flower ASA Allium sativum (ASA) from garlic Mannose Con-A Canavaliaensiformis (Con-A) from jack α-D-Mannose, α-D-Glucose, branched beanmannose GS-II Griffonia simplicoflia (GS-II) from shrub Terminal α- orβ-N-Acetylglucosamine GS HHA Hippeastrum hybrid (HHA) from Mannonse (intand term residues) amaryllis IRA Iris hybric (IRA) from Dutch IrisN-Acetyl-D-Galactosamine LEA Lycopersicon esculentum (LEA) fromβ(1,4)-linked N-Acetylglucosamine tomato LPA Limulus polyphemus (LPA)from Sialic Acid (N-Acetylneuraminic acid) horseshoe crab MIA Mangideraindica (MIA) from mango Exact specificity unknown PAA Perseau americana(PAA) from avocado Exact specificity unknown WGA Triticum vulgaris (WGA)from Wheat (GlcNAc-β-(1,4)-GlcNAc)1-4>β- Germ GlcNAc>Neu5Ac WGA-SSuccinyl Triticum vulgare (WGA-S) (GlcNAc-β-(1,4)-GlcNAc)1-4>β- fromwheat germ GlcNAc>Neu5Ac

Capture moieties suitable for use in methods of the invention may alsoinclude a nucleic acid ligand (aptamer). A nucleic acid ligand (aptamer)is a nucleic acid macromolecule (e.g., DNA or RNA) that binds tightly toa specific molecular target. Like all nucleic acids, a particularnucleic acid ligand may be described by a linear sequence of nucleotides(A, U, T, C and G), typically 15-40 nucleotides long. In solution, thechain of nucleotides forms intramolecular interactions that fold themolecule into a complex three-dimensional shape. The shape of thenucleic acid ligand allows it to bind tightly against the surface of itstarget molecule. In addition to exhibiting remarkable specificity,nucleic acid ligands generally bind their targets with very highaffinity, e.g., the majority of anti-protein nucleic acid ligands haveequilibrium dissociation constants in the picomolar to low nanomolarrange.

Nucleic acid ligands are generally discovered using an in vitroselection process referred to as SELEX (Systematic Evolution of Ligandsby EXponential enrichment). See for example Gold et al. (U.S. Pat. No.5,270,163). SELEX is an iterative process used to identify a nucleicacid ligand to a chosen molecular target from a large pool of nucleicacids. The process relies on standard molecular biological techniques,using multiple rounds of selection, partitioning, and amplification ofnucleic acid ligands to resolve the nucleic acid ligands with thehighest affinity for a target molecule.

In addition, the capture moiety may be a nucleic acid probe, typicallyan oligonucleotide, that specifically binds to a target nucleic acidsequence. A probe is generally a single-stranded nucleic acid sequencecomplementary to some degree to a nucleic acid sequence sought to bedetected (“target sequence”). A probe may be labeled with a reportergroup moiety such as a radioisotope, a fluorescent or chemiluminescentmoiety, or with an enzyme or other ligand which can be used fordetection. Standard techniques are used for nucleic acid and peptidesynthesis. These molecular biological techniques may be performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.,). Synthesis of complementary DNA is describedin, for example, Nature Methods 2, 151-152 (2005)doi:10.1038/nmeth0205-151. Background descriptions of the use of nucleicacid hybridization to detect particular nucleic acid sequences are givenin Kohne, U.S. Pat. No. 4,851,330 issued Jul. 25, 1989, and by Hogan etal., International Patent Application No. PCT/US87/03009, entitled“Nucleic Acid Probes for Detection and/or Quantitation of Non-ViralOrganisms,” both references hereby incorporated by reference herein.Hogan et al., supra, describe methods for determining the presence of anon-viral organism or a group of non-viral organisms in a sample (e.g.,sputum, urine, blood and tissue sections, food, soil and water).

Reporter phages may also be used as a capture moiety. Reporter phages,such as bacteriophages, are typically genetically modified phages usedto introduce a gene of interest into a host pathogen. The reporter geneincorporates detectable codes for a fluorescent or substrate dependentcolorimetric marker into host pathogen, which allows for subsequentpathogen detection. Bacteriophages used for pathogen detection aredescribed in detail in Singh, Amit, et al. “Bacteriophage based probesfor pathogen detection.” Analyst 137.15 (2012): 3405-3421; Dover, JasonE., et al. “Recent advances in peptide probe-based biosensors fordetection of infectious agents.” Journal of microbiological methods 78.1(2009): 10-19.

Methods for attaching the target-specific binding moiety to the magneticparticle are known in the art. Coating magnetic particles withantibodies is well known in the art, see for example Harlow et al.(Antibodies, Cold Spring Harbor Laboratory, 1988), Hunter et al.(Immunoassays for Clinical Chemistry, pp. 147-162, eds., ChurchillLivingston, Edinborough, 1983), and Stanley (Essentials in Immunologyand Serology, Delmar, pp. 152-153, 2002). Such methodology can easily bemodified by one of skill in the art to bind other types oftarget-specific binding moieties to the magnetic particles. Certaintypes of magnetic particles coated with a functional moiety arecommercially available from Sigma-Aldrich (St. Louis, Mo.).

Since each set of particles is conjugated with capture moieties havingdifferent specificities for different pathogens, compositions used inmethods of the invention may be provided such that each set of capturemoiety conjugated particles is present at a concentration designed fordetection of a specific pathogen in the sample. In certain embodiments,all of the sets are provided at the same concentration. Alternatively,the sets are provided at different concentrations. For example,compositions may be designed such that sets that bind gram positivebacteria are added to the sample at a concentration of 2×10⁹ particlesper/ml, while sets that bind gram negative bacteria are added to thesample at a concentration of 4×10⁹ particles per/ml. Compositions usedwith methods of the invention are not affected by antibodycross-reactivity. However, in certain embodiments, sets are specificallydesigned such that there is no cross-reactivity between differentantibodies and different sets.

The sets of magnetic particles may be mixed together to isolate certainfungi, bacteria, or both. These sets can be mixed together to isolatefor example, E. coli and Listeria; or E. coli, Listeria, andClostridium; or Mycobacterium, Campylobacter, Bacillus, Salmonella, andStaphylococcus, etc. One set may be specific to a certain bacterium andanother set may be specific to a certain fungus. Any combination of setsmay be used and compositions of the invention will vary depending on thesuspected pathogen or pathogens to be isolated. In certain embodiments,compositions include two, three, four, five . . . 10, 20, etc. differentsets of magnetic particles conjugated to different pathogens.

In preferred embodiments, sets of magnetic particles conjugated tocapture moieties that are specific to different targets withinmulti-plex detection panel. For example, sets of modified magneticparticles may be chosen to isolate two or more certain pathogens. Thetwo or more certain pathogens chosen may be causal of similar symptoms(e.g. digestive abnormalities), commonly found in the type of body fluid(e.g. stool), or pathogens common to a certain area (e.g. hospitalsetting). The following tables show exemplary panels of fungal (Table 2)targets and bacterial (Table 3) targets. “Sp” means a target specieswithin a genus, and “Sp” means a target.

TABLE 2 Fungal Panels-Compositions of the invention will include capturemoieties bound to magnetic particles specific to each of the targets inthe panel. Fungi Assay Target 1 Target 2 Target 3 Target 4 Target 5Panel A Species of C. albicans C. glabrata C. parapsilosis N/A Candida(or C. tropicalis) genus Panel B Species of C. albicans C. glabrata C.parapsilosis/ C. krusei Candida C. tropicalis genus Panel C Species ofC. albicans C. glabrata/ C. parapsilosis/ Cryptococcus Candida C. kruseiC. tropicalis spp. genus Panel D Candida C. albicans/C. parapsilosis/ C.glabrata/C. krusei Aspergillus spp. Cryptococcus genus C. tropicalisspp. Panel E Cryptococcus C. albicans/C. parapsilosis/ C. glabrata/C.krusei Aspergillus spp. Cryptococcus neoformans C. tropicalis gattii

TABLE 3 Bacteria Panels-Compositions of the invention will includecapture moieties bound to magnetic particles specific to each of thetargets in the panel. Bacteria Assay Target 1 Target 2 Target 3 Target 4Target 5 Panel S. aureus CoNS mecA Streptococcus N/A A-sp./Enterococcocus Gram sp. Positive Panel Panel EnterobacteriaceaePseudomonas Acinetobacter Stenotrophomonas N/A B- aeruginosa sp.maltophilia Gram (or other GNR Negative such as Panel Neisseria,Haemophilus) Panel C Staphylococcus CoNS Enterobacteriaceae PseudomonasN/A sp. aeruginosa Panel D Staphylococcus Enterococcus E. coliPseudomonas N/A sp. sp./Streptococcus aeruginosa sp. Panel EStaphylococcus CoNS Enterococcus Enterobacteriaceae N/A aureussp./Streptococcus sp. Panel F Staphylococcus EnterococcusEnterobacteriaceae Candida spp. N/A sp. sp./Streptococcus sp. Panel S.aureus CoNS mecA Streptococcus Enterococcocus G- sp. sp. Gram PositivePanel Panel Enterobacteriaceae Pseudomonas AcinetobacterStenotrophomonas Neisseria H- aeruginosa sp. maltophilia meningitidisGram Negative Panel Panel I Staphylococcus Enterococcus StreptococcusEnterobacteriaceae Pseudomonas sp. sp. sp. aeruginosa Panel JStaphylococcus Enterococcus Streptococcus E. coli Pseudomonas sp. sp.sp. aeruginosa Panel K Staphylococcus CoNS EnterococcusEnterobacteriaceae Pseudomonas aureus sp./Streptococcus aeruginosa sp.Panel L Staphylococcus Enterococcus Enterobacteriaceae PseudomonasAcinetobacter sp. sp./Streptococcus aeruginosa sp. sp.

In certain embodiments, compositions of the invention include at leastone set magnetic particles conjugated to a capture moiety specific to aninternal control (IC). For example, an IC detectable marker may beplaced into a clinical sample suspected of containing a pathogen. Fordetection of the pathogen, a plurality of sets of magnetic particles areintroduced to that clinical sample, in which at least one set isconjugated to a capture moiety designed to bind the IC detectable markerand one or more other sets are conjugated to capture moieties designedto bind to the suspected pathogens. When the assay is conducted tocapture and isolate the pathogen, the presence or absence of the ICdetectable marker indicates whether the assay properly separated themarker from the sample. That is, the introduction of a detectable markerallows one to determine whether the sets of magnetic particlesconjugated to capture moieties are properly capturing or isolating thetarget pathogens that are present within the fluid. This is important todistinguish between a failed assay (i.e. failure to identify a targetpathogen present in the sample) and a positive assay (i.e. positivelydetermining the absence of a target pathogen). The presence of themarker indicates that the assay worked properly; and thus any detectionof pathogen (or the absence of the pathogen) is accurate and not theresult of a failed assay. Use of IC detectable markers is described inmore detail in co-owned U.S. provisional application No. 61/739,577,filed Dec. 18, 2012. The panels listed in Tables 2 and 3 above mayinclude the addition of an IC detectable marker inserted into thesample, and the sets of magnetic particles would include sets conjugatedto capture moieties specific to targets listed in the panel as well asthe internal control.

Methods of the invention that utilize the internal control involveobtaining a sample suspected of containing a pathogen and introducing adetectable maker into the sample. An assay is conducted to detect thesuspected pathogen and the detectable marker in the sample using aplurality of sets of magnetic particles (as described herein). Membersof at least one set of magnetic particles are conjugated to bindingentity specific to a pathogen, and members of at least one set ofmagnetic particles are conjugated to a binding entity specific to thedetectable marker. After the conducting step, the presence of absence ofthe marker in the sample is determined. Based on the presence or absenceof the detectable marker, a determination is made about the presence orabsence of targets in the sample. The presence of the marker indicatesthat the assay worked properly; and thus any detection of pathogen (orthe absence of pathogen) is accurate and not the result of a failedassay.

Any detectable marker may be used for an internal control. In certainembodiments, the IC detectable marker may be a microbe, including aviable or nonviable microbe. In addition, the IC detectable marker canbe sufficiently similar to the target such that the assay performsfunctionally in the same manner for the detectable marker and thetarget. The detectable marker may be labeled or otherwise modified toallow for their differentiation from targets originally present in thesample. For example, but not by way of limitation, the detectable markercan be genetically modified so as to express a fluorescent protein, oralternatively, the detectable marker could be pre-stained with apersistent stain to allow their differentiation from microbes that areoriginally present in the fluid composition. In addition, the detectablemarker may be chosen based a chromogen dye specific reaction to thepresence of the detectable marker.

Reagents and Buffers

The target capture system can employ reagents and buffers for carryingthe processes of the target capture system. In certain aspects, thecartridge of the target capture system includes reservoirs for storingthe reagents and buffers. The cartridge also includes components such aschannels, valves, ect. that provide a means for delivering the reagentand buffers within the cartridge. Accordingly, each of the reagents,buffers, and fluids described below can be stored within the cartridgeand delivered into the sample to carry out the various processes of thetarget capture system.

In certain embodiments, a buffer solution is added to the sample alongwith the magnetic particles to facilitate binding of the particles totargets within the sample. The buffer can be stored within a reagentreservoir within the cartridge and introduced to the sample duringprocessing. An exemplary buffer includesTris(hydroximethyl)-aminomethane hydrochloride at a concentration ofabout 75 mM. It has been found that the buffer composition, mixingparameters (speed, type of mixing, such as rotation, shaking etc., andtemperature) influence binding. It is important to maintain osmolalityof the final solution (e.g., blood+buffer) to maintain high labelefficiency. In certain embodiments, buffers used in devices and methodsof the invention are designed to prevent lysis of blood cells,facilitate efficient binding of targets with magnetic beads and toreduce formation of bead aggregates. It has been found that the buffersolution containing 300 mM NaCl, 75 mM Tris-HCl pH 8.0 and 0.1% Tween 20meets these design goals.

Without being limited by any particular theory or mechanism of action,it is believed that sodium chloride is mainly responsible formaintaining osmolality of the solution and for the reduction ofnon-specific binding of magnetic bead through ionic interaction.Tris(hydroximethyl)-aminomethane hydrochloride is a well-establishedbuffer compound frequently used in biology to maintain pH of a solution.It has been found that 75 mM concentration is beneficial and sufficientfor high binding efficiency. Likewise, Tween 20 is widely used as a milddetergent to decrease nonspecific attachment due to hydrophobicinteractions. Various assays use Tween 20 at concentrations ranging from0.01% to 1%. The 0.1% concentration appears to be optimal for theefficient labeling of bacteria, while maintaining blood cells intact.

Additionally, devices and methods of the invention employ wash solutionsto reduce particle aggregation and remove unwanted sample, non-specifictarget entities, and buffer. Exemplary solutions include heparin,Tris-HCl, Tris-borate-EDTA (TBE), Tris-acetate-EDTA (TAE),Tris-cacodylate, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulphonicacid), PBS (phosphate buffered saline), PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid), MES(2-N-morpholino)ethanesulfonic acid), Tricine(N-(Tri(hydroximethyl)methyl)glycine), and similar buffering agents. Inparticular embodiments, the wash solution includes heparin. Forembodiments in which the body fluid sample is blood, the heparin alsoreduces probability of clotting of blood components after magneticcapture. These wash solutions can be contained in one or more reagentreservoirs of the cartridge and are typically introduced into a magnetictrap flow through chamber during separation of the magnetic particles.

In certain embodiments, the target capture system includes introducing asolution to invoke cell lysis in capture target cells in order torelease the nucleic acid. The solution can be any suitable lysisfluid/buffer capable of lysing the cells and/or particles of interest inthe fluid sample. An example of a suitable lysis buffer is 100 mMTris/HCl, 8 M GuSCN (pH 6.4).

In addition, the target capture system may also utilize an elutant fluidto elute purified nucleic acids from a nucleic acid extraction column.The eluant fluid can be any fluid suitable for eluting purified nucleicacids from the nucleic acid extraction unit. Examples of suitableelution fluids include water and 10 mM Tris/HCl, 1 mM EDTA Na.sub.2 (pH8).

In addition, a reagent can be used that disrupts the interaction betweenthe particle and the target cell, e.g. disrupts an antibody-antigenreaction. This reagent may be used after capture of the target/magneticparticle complexes to separate isolated whole target cells from themagnetic particles.

Cartridge

The target capture system of the invention includes a cartridge that isa single structure having one or more components (such as reagentreservoirs, magnetic traps, storage reservoirs, flow chambers, etc.)that are formed within the cartridge. These components can be connectedvia channels formed within the system. As such, there is no need forexternal tubing or other external attachments to connect the componentsof the cartridge.

A significant advantage of certain embodiments is that the cartridgeincludes both macrofluidic and microfluidic components and can processmacrofluidic and microfluidic volumes of fluids to isolate a target.This aspect of the invention accounts for the fact that a minute amountof targets (such as pathogens) may be present in a sample having amacrofluidic volume which necessitates processing the entiremacrofluidic volume in order to increase the likelihood that the targetwill be isolated. To isolate targets in a microfluidic device, theentire macrofluidic volume of sample would have to be transferred slowlyor in a piecemeal fashion (e.g. via pipetting) into a microfluidicdevice at microfluidic rate, which undesirably takes a long amount oftime and risks losing the target analyte of interest during thetransfer. In certain aspects, the cartridge is designed to consolidate asample of macrofluidic volume into a concentrated microfluidic volume offluid that contains target cells of interest. The concentratedmicrofluidic volume is then processed at the microfluidic level.

Generally, microfluidics relates to small sample volumes and smallchannel pathways. For example, microfluidic volumes are normally below 1mL, or on the microliter (μL) scale or smaller, for example, nL(nanoliters) and pL (picoliters). As used herein, microfluidic volumesrelate to volumes less than 1 mL. In addition, microfluidics relates tosmall channel pathways on the micrometer scale. As used herein,microfluidic channels within systems of the invention refer to channelsthat have channel heights and/or widths equal to or less than 500 μm.See “Microfluidics and Nanofluidics: Theory and Selected Applications,”Kleinstruer, C., John Wiley & Sons, 2013, which is incorporated byreference. The channel height or width is defined as the height or widthof the path that the sample volume must pass through within thecartridge. Comparatively, macrofluidics volumes relate to volumesgreater than the microliter (μL) scale, for example, sample volumes onthe milliliter (mL) scale. As used herein, macrofluidic volumes arevolumes of 1 mL or greater. Macrofluidic channels within systems of theinvention are channels having channel heights and/or widths of greaterthan 500 μm.

Other macrofluidic components are chambers, reservoirs, traps, mixers,etc. Such macrofluidic components are dimensioned to hold 1 mL or moreof fluid. For example, the individual volume can range withoutlimitation from about 10 to about 50 mL. Other microfluidic componentsare chambers, reservoirs, traps, mixers, etc. Such microfluidiccomponents are dimensioned to hold less than 1 mL of fluid. For example,the individual volumes can range without limitation from about 1 μL toabout 500 μL.

The cartridge includes channels to facilitate transportation ofsubstances and fluids through into, within, and out of the cartridge.The channel generally will include characteristics that facilitatecontrol over fluid transport, e.g., structural characteristics (anelongated indentation) and/or physical or chemical characteristics (e.g.lined with a solution or substance that prevents or reduces adherenceaggregation of sample/particulates) and/or other characteristics thatcan exert a force (e.g., a containing force) on a sample or fluid. Thechannels can be independent, connected, and/or networked betweencomponents of the cartridge. Some (or all) of the channels may be of aparticular size or less, for example, having a dimension perpendicularto the flow of fluid to achieve a desired fluid flow rate out of onecomponent and into another. The channels can be designed to transfermacro and micro scales of fluid.

The channels of the cartridge can connect to and interconnect thecomponents of the cartridge. The cartridge can include one or more ofthe following components: through holes, slides, foil caps, alignmentfeatures, liquid and lyophilized reagent storage chambers, reagentrelease chambers, pumps, metering chambers, lyophilized cakereconstitution chambers, ultrasonic chambers, joining and mixingchambers, mixing elements such as a mixing paddle and other mixing gear,membrane regions, filtration regions, venting elements, heatingelements, magnetic traps/chambers, reaction chambers, waste chambers,membrane regions, thermal transfer regions, anodes, cathodes, anddetection regions, drives, plugs, piercing blades, valve lines, valvestructures, assembly features such as o-rings, instrument interfaceregions, cartridge/vessel interfaces, one or more needles associatedwith the sample interface, optical windows, thermal windows, anddetection regions. These components can have macro- or micro-volumes.

The cartridge includes at least one inlet for introducing sample intothe cartridge and at least one inlet for allowing the instrument tointroduce air pressure, e.g., to drive fluid flow, or to introducefluids into the cartridge. The cartridge further includes at least oneoutlet to deliver a final product to the operator, e.g. a capturedtarget or nucleic acids of a captured target into a removable vial forfurther analysis. In preferred embodiments, the inlet and outlet areassociated with the cartridge/vessel interface of the inventiondescribed in detail hereinafter.

In one embodiment, the cartridge further includes sensing elements todetermine the stage of the processes performed within the cartridge. Thesensing elements can be used to gauge the flow within the cartridge andthe timing for when certain subsystems of the instrument interact withthe cartridge. The sensing elements include, but are not limited to,optical sensors (e.g. for monitoring the stage of processing within thechamber), timers (e.g., for determining how long a sample is in a mixingchamber or in a reaction chamber); air displacement sensors (e.g. fordetermining the volume of fluid within one or more chambers);temperature sensors (e.g. for determining the temperature of areaction), bubble sensor (e.g. for detecting air and/or volume of fluidwithin chambers and fluid flow; pressure sensors for determining, e.g.,rate of fluid flow.

In certain aspects, fluids and substances are driven into, within, andout the cartridge via one or more drive mechanisms. The drive mechanismscan be located on the cartridge itself or located on an instrument incombination with the cartridge. The drive mechanisms provide a means forfluid control within the cartridge and allows for transport of fluid andsubstances within the cartridge. In addition, the drive mechanismsprovide a means for transferring fluids and substances between thecartridge and the vessel at the cartridge/vessel interface. In oneembodiment, the drive mechanism is a part of the instrument and isoperably associated with the cartridge at one or morecartridge/instrument interface. The cartridge can include a filter atthe cartridge/instrument interface to prevent unwanted particles fromentering the cartridge from the drive mechanism or instrument. Thefilter also prevents sample and other fluids from exiting the cartridgeat the cartridge/instrument interface. The drive mechanisms of theinstrument are discussed in more detail hereinafter.

The cartridge (whether including macrofluidic components, microfluidiccomponents, or both) can be fabricated using a variety of methods,including without limitation, computer numerical control (CNC)techniques, traditional lithographic techniques, soft lithography,laminate technologies, hot embossing patterns, die cutting, polymermolding, combinations thereof, etc. The cartridge can be fabricated fromany etchable, machinable or moldable substrate. The term machining asused herein includes, without limitation printing, stamping cutting andlaser ablating.

Suitable materials for the cartridge include but are not limited tonon-elastomeric polymers, elastomeric polymers, fiberglass, Teflon,polystyrene and co-polymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polycarbonate, polyurethanes,TEFLON (polytetrafluoroethylene, commercially available by the DuPontcompany), and derivatives thereof. Preferably, the cartridge and thecartridge components are formed primarily from plastic. Plastics arecost-efficient and allow for the cartridge to be economicallymanufactured at a large scale. As such, the cartridge can be designed asa single use, disposable cartridge.

There are some components of the cartridge that are not plastic, andthese components can be formed from, for example, metals, silicon,quartz, and glass. These components include but are not limited tosurfaces, glass ampoules, filters, assembly materials (such as screwsand other fasteners), electrode pins, membrane, affinity columns, andcollection vials.

The cartridge can also include thin film layers that formstructures/interfaces (such as walls and valves) on the cartridge,interfaces between components within the cartridge, and interfacesbetween the cartridge and the instrument. In one aspect, the thin filmlayers are for bonding fabricated components together (such as CNCcomponents and lithographic components), sealing components together,providing conduits between components, transferring stimulation betweencomponents (e.g. capable of transferring physical or mechanicalstimulation from an assembly/system on the instrument to a chamber inthe cartridge), supporting elements, covering the channel, functioningas a cap and/or frangible seal for reservoirs or chambers, andperforming as a valve. The thin film can be elastomeric ornon-elastomeric material. In certain aspects, the thin film is a polymeror thermoplastic polymer. Exemplary polymers or thermoplastic polymerscan include, but are not limited to, polymers selected from the groupconsisting of polymethyl methacrylate (PMMA), polycarbonate (PC),polyvinylacetate (PVAc), polystyrene (PS), polypropylene, polyethylene,polymethyl methacrylate, poly(amides), poly(butylene), poly(pentadiene),polyvinyl chloride, polycarbonate, polybutylene terephthalate,polysulfone, polyimide, cellulose, cellulose acetate, ethylene-propylenecopolymer, ethylene-butene-propylene terpolymer, polyoxazoline,polyethylene oxide, polypropylene oxide, polyvinylpyrrolidone, andcombinations thereof. In addition, thin film can be an elastomer,polymer blend and copolymer selected from the group consisting ofpoly-dimethylsiloxane (PDMS), poly(isoprene), poly(butadiene), andcombinations thereof. In some embodiments, the thin film includes rubber(including silicone) alone or in combination with a polymer.

In a preferred embodiment, the cartridge is pre-assembled prior toshipment to distributors/customers. The pre-assembled cartridge may alsoinclude one or more of reagents, capture particles (including magneticparticles), lysing beads, water, and other substances/fluids pre-loadedinto one or more chambers or reservoirs formed within the cartridge. Thepre-assembled cartridge may be partially pre-loaded, e.g. loaded withonly a portion of the components necessary to isolate a target. Ifpre-assembled, the cartridge can include reagents and magnetic particlesspecific certain isolation assays and/or specific to certain targetanalytes. In addition, the cartridge can include magnetic particles withbinding moieties specific to a plurality of different targets to providefor isolation of a target when the suspected target is not known. Seeco-pending and co-assigned U.S. application Ser. No. 13/091,506 thatdescribes compositions for isolating a target sample from aheterogeneous sample. It is also contemplated that the cartridge ispartially-assembled prior to shipment to distributors/customers to allowthe individual customer to load the cartridge with reagents, beads, ect.that are tailored to the analysis/identification needs of the customer.

FIG. 2 depicts a schematic overview of a cartridge 100 according to theinvention. FIG. 3 depicts the various chambers as described in theschematic overview of the cartridge in FIG. 2. The cartridge 100 asdepicted in FIG. 2 includes both macrofluidic and microfluidiccomponents. However, it is understood that the cartridge could beconstructed with only macrofluidic or microfluidic components. Channels,valves, and other structures are included in the cartridge to facilitateand control communication between cartridge components.

As shown in FIG. 2, the cartridge 100 includes inlet port 140 a andoutlet port 150 a. In inlet port 140 a can introduce sample or otherfluids into the cartridge and outlet port 150 a allows for transfer offluid/substances/pressure out of the cartridge and into a vessel 10containing sample. Preferably, the inlet port 140 a and outlet port 150a corresponds to the cartridge/vessel interface, which includes inletmember 140 and outlet member 150 (not shown). Inlet members 140 andoutlet member 150 extend to into the vessel 10 containing sample coupledto the cartridge. The outlet port 150 a allows fluids/substances to betransferred out of the cartridge 100 and into a sample vessel 10. Theinlet port allows transfer of sample/fluids/substances from the vessel10 to the cartridge 100. The inlet port 140 a and outlet port 150 a areassociated with channels within the cartridge that direct fluid flow andcan also be associated with one or more valves. The valves can be usedto start, stop, increase, and/or decrease fluid into the inlet port 140a and out of the outlet port 150 a.

Along the top of the cartridge 100 are several drive ports 190 at theinstrument interface 45. The drive ports 190 connect various features onthe cartridge 100 to the instrument's 200 drive mechanism. As shown inFIG. 2, a port 190 is in communication with a particle buffer reservoir20. The particle buffer reservoir 20 can contain a buffer that promotesbinding of particles and targets. The particle buffer reservoir 20 is incommunication with a particle chamber 40 containing a plurality ofmagnetic particles. The plurality of magnetic particles may be used toisolated one or more targets of interest. As discussed above, theplurality of magnetic particles may include different sets of magneticparticles in which each set is conjugated to capture moiety specific toa different target of interest (e.g. for multiplex capture of targets).The particle chamber 40 is typically a glass ampoule. Glass ampoules areideal because they are able to keep the magnetic particles in a constantenvironment (e.g. of a certain air pressure and/or humidity). Theplurality of particles within the particle chamber 40 can includemoieties specific to a target. The particle buffer reservoir 20 and theparticle chamber 40 are in communication with the outlet port 150 a. Theparticles and buffer can be transported by the drive mechanism out ofthe outlet port 150 a and directly into the vessel 10 containing asample.

The inlet port 140 a is connected to a channel that directs the contentsof the vessel (sample/particle/buffer) into a mixing chamber 170. Themixing chamber 70 can be used to incubate and agitate thesample/particle/buffer mixture. The mixing chamber 70 can include amixing paddle disposed therein. A portion of the mixing paddle isoperably associated with a mechanical drive on the instrument thatcauses the mixing paddle to move or rotate within the mixing chamber 70.

The mixing chamber 170 is in communication with a first magnetic trap 60and a magnetic trap overflow 50. The first magnetic trap 60, as shown inFIG. 2, is a flow through chamber in which fluid can be driven into andout of the first magnetic trap 60. The first magnetic trap 60 can be aflow chamber having a planar or orthogonal inlet. In one embodiment, thefirst magnetic trap 60 has an orthogonal inlet because this inlet shapecreates a uniform flow profile through the magnetic trap. The magnetictrap 60 is configured to engage with a magnet assembly of the instrument200 to separate particles from the sample/buffer via application of amagnetic field. The magnetic assembly can include an array of barmagnets which are perpendicular to a flow of fluid within the firstmagnetic trap 60 and have a magnetic field sufficient to force themagnetic particles against a surface of the magnetic trap, therebyseparating the magnetic particles. The surface of the first magnetictrap 60 may include binding moieties specific to the magnetic particlesthat, in addition to the magnetic field, aid in separating magneticparticles from the sample/buffer solution. The magnetic assembly thatinteracts with the first magnetic trap 60 may also include a swipermagnet that generates a magnetic field to release the magnets from thesurface of the magnetic trap 60 after separation. The magnetic trapoverflow chamber 50 is for receiving and transferringbuffer/particle/sample solution to and from the first magnetic trap 60.

As further shown in FIG. 2, the first magnetic trap 60 is incommunication with a wash buffer reservoir 35. A wash buffer dripchamber 62 between the first magnet trap 60 and the wash bufferreservoir 35 can control flow of buffer into the first magnetic trap 60.The wash buffer reservoir 35 may contain buffer to rinse extrasample/buffer from magnetic particles isolated in the first magnetictrap 60. In addition, the wash buffer can be used to transfer theseparated magnetic particles to the second magnetic trap 70.

Optionally, a pre-magnetic trap chamber 82 is between the first magnetictrap 60 and the second magnetic trap 80 as shown in FIG. 2. Thepre-magnetic trap chamber 82 can be used to control flow of particlesand wash buffer from the first magnetic trap 60 to the second magnetictrap 80. In certain aspects, the first magnetic trap 60 is macrofluidicand the second magnetic trap 80 is microfluidic. In such aspect, thepre-magnetic trap chamber 82 acts an intermediate component to aid intransitioning from macro-to-micro by controlling fluid flow from thefirst magnetic trap 60 and the second magnetic trap 80.

The second magnetic trap 80 is configured to engage with a magnet of theinstrument to further separate any remaining sample/buffer from themagnetic particles. The second magnetic trap 80 is a flow throughchamber in communication with a second magnetic trap overflow chamber105. The second magnetic trap overflow chamber 105 is used to storeunwanted buffer/sample (waste) from the second magnetic trap 80. Thesecond magnetic trap 80 is in communication with a lysis bufferreservoir 90 and optionally, a lysis buffer drip chamber 107 to controlflow of lysis buffer into the second magnetic trap 80. The secondmagnetic trap 80 is also configured to engage with a sonication deviceof the instrument. In one embodiment, a wall of the second magnetic trap80 that interfaces with the instrument has a certain thickness, such as125 μM, that allows vibrations of the sonication device to invoke celllysis on targets within the second magnetic trap. The wall interfacingthe sonication device can be a Mylar film. The second magnetic trap 80can optionally include binding moieties specific to the magneticparticles to assist in isolating the magnetic particles.

The second magnetic trap 80 is also in communication with a pre-columnmixer 85, which receives the lysate from the second magnetic trap 80.The pre-column mixer 85 is in communication with to a nucleic acidbinding buffer reservoir and in communication with a nucleic acidextraction column 110. An output chamber 95 can be included between thepre-column mixer 85 and the nucleic acid extraction member 110. Anynucleic acid extraction member 110 that retains extracted nucleic acidwhile allowing the other fluids such as lysis debris to flow through themember is suitable for use in the invention. The nucleic acid extractionmember 110 can be a filter or a column, such as an affinity column.Examples of nucleic acid extraction members are described in, forexample, United States Patent Publication No. 2011/0300609.

One or more column wash reservoirs 65 are connected to the nucleic acidextraction member 110 to direct unwanted sample/buffer/ect. from thecolumn 110 to a waste reservoir. An elution reservoir 55 contains abuffer or fluid that is capable of eluting nucleic acids disposed withinthe nucleic acid extraction member. The fluid, such as water, is flushedfrom the elution reservoir 55 through extraction member 110 to elutepurified nucleic acids into a collection vial (not shown).

In one embodiment, the particle chamber 40, the wash buffer 35, themixing chamber 70, the first magnetic trap 60 and the magnetic trapoverflow 50 of the cartridge 100 are all macrofluidic componentsdesigned to process a macrofluidic volume of fluid. Because thesecomponents are macrofluidic, the entire sample can be subject to theincubation, agitation, and the first magnetic separation step. After themagnetic particles are isolated in the first magnetic trap 60, a washbuffer flows through the first magnetic trap 60 to transport theseparated particles to the second magnetic trap 80. The cartridge 100components after the first magnetic trap 60 are microfluidic, includingthe second magnetic trap 60, magnetic trap overflow 105, pre-columnmixer 80. The second magnetic trap 80 isolates substantially the entirequantity of magnetic particles within a microfluidic volume of fluidfrom the macrofluidic volume of fluid. The rate of fluid flow betweenthe first and second magnetic traps can be adjusted to allow for thesecond magnetic trap 80 to isolate all of the magnetic particles. Thus,the macrofluidic volume of sample is concentrated into a microfluidicvolume of concentrated clinically relevant sample. The concentratedmicrofluidic volume of fluid allows for more efficient nucleic acidextraction.

Cartridge and Vessel Interface

For isolation and detection assays conducted on cartridges or chips(whether microfluidic or macrofluidic), it is important to transfer theentire obtained sample from a collection device into the cartridge toincrease the efficiency of isolation or detection. Especially insituations where there is little sample, which is often the case inforensic analysis, or when there is a small concentration of targets permL of sample (e.g. 1 CFU/mL), which is often the case for pathogenicdetection. Cartridges of the invention include a cartridge/vesselinterface designed to maximize the amount of sample transferred into thevessel and the amount of sample subject to the cartridge processes toavoid loss of clinically relevant within a sample collection deviceduring sample transfer. It is understood that the cartridge/vesselinterface can be included on the cartridge of the target capture systemand any other cartridge for processing a sample.

The cartridge/vessel interface may include one or more input and/oroutput members that enter a vessel containing sample to maximize theamount of sample that is transferred from the vessel containing sampleinto the cartridge for processing. In one embodiment, thecartridge/sample interface includes an inlet member and an outlet memberto facilitate communication of fluids and substances out of thecartridge and into the sample vessel and to facilitate communication offluids and substances (including the sample) out of the vessel and intothe cartridge. The outlet member also provides for 1) introducing air toforce the sample into the cartridge via the inlet port and/or the inletmember to maximize drainage; 2) introducing a fluid into the vessel torinse the vessel container to transfer any remaining sample in thevessel into the cartridge; and 3) introducing fluids/substancesnecessary for cartridge processes directly to the entire sample toensure the entirety of the sample engages with those fluids/substances.The input member provides for transferring the vessel contents into thecartridge for processing.

In certain embodiments, the fluid is introduced into the vessel at thesame time the vessel contents (including sample and/or fluid) istransferred into the cartridge. Alternatively, the sample is at leastpartially transferred from the vessel into the cartridge prior tointroducing the fluid from the cartridge into the vessel.

In one embodiment, both the inlet member and outlet member define alumen and include a penetrating tip. For example, the inlet member andthe outlet member can be hollow pins or needles. The inlet member andoutlet member correspond with inlet and outlet ports on the cartridge.The input member and output member are designed to penetrate the vesselcontaining the sample to place the vessel (and thus the sample) incommunication with the cartridge. The communication between the vesseland the cartridge through the input members and output members may befluidic, pneumatic, or both. The input and output members can also actto couple the vessel to the cartridge and maintain the position of thevessel on the interface.

In certain embodiments, the input and output members are incommunication with a drive mechanism. The drive mechanism can be a partof the cartridge itself or located on an instrument for use with thecartridge. The drive mechanism can apply air pressure or a vacuum forceto facilitate transportation between the vessel and cartridge. Forexample, the drive mechanism can apply air pressure through a channel ofthe cartridge, out of the output member, and into the vessel to forcethe vessel contents to drain through the input member. In addition, thedrive mechanism can apply a vacuum force to the input member to forcethe sample to drain into member.

The input member is in communication with one or more components of thecartridge (e.g. a mixing chamber, magnetic trap, storage reservoir,reagent reservoirs, etc.) that process fluids delivered from the vesselinto the cartridge. The input member allows for fluids to transfer outof the vessel and into the cartridge for processing.

The output member is in communication with one or more components of thecartridge (e.g. storage reservoir, reagent reservoir, magnetic trap,etc.) to allow delivery of fluids, substances, and/or gases from thecartridge into the vessel container. In one embodiment, fluid from areagent reservoir is driven through the output member and into thevessel to rinse sides of the vessel. The fluid may contain one or moresubstances. In one aspect, the fluid includes capture particles havingbinding moieties specific to one or more suspected targets within thesample. The fluid can be any fluid that does not interfere with theprocesses of the cartridge. In another embodiment, the fluid is anessential element of the cartridge processes. For example, the fluid canbe a buffer that promotes a reaction within the sample, such aspromoting target capture. In addition, fluids, substances, and/or gasesmay be subject to a reaction/process within the cartridge prior to beingdelivered into the vessel. For example, a buffer may be heated in thecartridge prior to introducing the buffer into the vessel.

In certain embodiments, one or more input members and one or more outputmembers are inserted into the vessel to place the vessel incommunication with the cartridge. This allows, for example, the vesselcontents to be directed through one or more input member into one ormore different channels in the cartridge for processing. In addition,one or more output members may be for delivering different fluids orreagents into the vessel.

The vessel for coupling to the cartridge interface can be an open orclosed container. In one embodiment, the vessel is a collection tube,such as a VACUTAINER (test tube specifically designed for venipuncture,commercially available from Becton, Dickinson and company). Ideally, thevessel is enclosed, such as a collection tube enclosed by a stopper or aplug. The stopper or plug can be rubber, silicone, or polymericmaterial. For coupling the vessel to the sample, the vessel or thevessel plug is pressed against the input and output member until theinput and output member are inserted into the vessel. The cartridgeinterface can also include a vessel holder to properly position thevessel onto the needles and a locking mechanism to lock the vessel inplace while coupled to the cartridge. These features provide a snug fitof the cartridge and the vessel.

Vessels suitable for use with the cartridge can be of any volume size.For example, the vessels can range in volumes from 0.1 to 1 mL to 100mL. In one embodiment, the vessel has a volume of 10 mL. The volume ofthe vessel may depend on the sample and the suspected target to bedetected. That is, the vessel should be of a sufficient volume tocontain an amount of sample fluid in which it is more likely than notthat a suspected target is present.

In an embodiment, the output member is positioned within the vessel sothat the output member delivers a fluid to the top of the vessel. Thiscauses the fluid to run down at least one side of the vessel and rinseany sample that may have collected along the side of the vessel. Thedrive mechanism can be set to apply a pressure sufficient to deliver thefluid out of the output member so that it hits the top surface of thevessel. In addition, the input member is positioned within the vessel topromote drainage of the vessel contents. For example, the input memberis level with or below the bottom of the vessel. In one embodiment, thevessel or the vessel plug is shaped to drain into the input member. Forexample, the vessel plug is conically-shaped.

In addition, the drive mechanism may provide sufficient pressure torelease capture particles out of the output member and into the vessel.For example, pressure from the drive mechanism releases a buffer into achamber having a plurality of capture particles disposed therein. Thebuffer/capture particles are then driven from the cartridge through theoutput member and directly into the vessel. The drive mechanismcontinues to force the buffer through the particle chamber and into thevessel until all of the capture particles are transferred into thevessel. At the same time, the input member may transfer the sample,buffer, and capture particles out of the vessel and into the cartridge.After substantially all the sample and capture particles are transferredinto the cartridge, fluid or the buffer can continue to be introducedinto the sample for an additional rinse. In another embodiment, theinput member transfers at least a portion of the sample into thecartridge prior to introduction of the capture particles/buffer toprovide space within the vessel.

FIG. 4 highlights the vessel/cartridge interface 120 of the cartridge100. The vessel/cartridge interface couples the vessel 10 to thecartridge 100 and provides communication (including fluidic andpneumatic communication) between the cartridge and the vessel. Thevessel/cartridge interface includes output member 150 and input member140. The output member 150 and input member 140 are hollow pins orneedles that penetrate the vessel 10 to place the vessel 10 incommunication with the cartridge 100. The input member 140 and outputmember 150 penetrate a stopper 410 coupled to the vessel. Thevessel/cartridge interface 120 can include guide 205. For loading, thevessel 10 is pushed through the guide 205 to direct and align the vessel10 onto the input member 140 and output member 150. One or morepositioning arms 210 are designed to hold the vessel 10 in place oncepositioned onto the input member 140 and output member 150. In addition,the cartridge can include a vessel cover 215 that closes over the vessel10 as coupled to the cartridge 100.

Instrument

In certain aspects, the cartridge interfaces with and is used inconjunction with an instrument. The instrument provides, for example,the pneumatic, fluidic, magnetic, mechanical, chemical functions, asnecessary to process the sample within the cartridge. In one aspect, thecartridge is inserted into the instrument for processing and theinstrument is turned on by an operator to activate sample processing.Once the cartridge is loaded into the instrument, the system does notrequire further manual technical operations on behalf of the operator.

In one embodiment, the instrument contains drive mechanisms that connectto the cartridge when inserted into the instrument. Any drive mechanismknown in the art may be used with target capture system, includingpneumatic drive mechanisms, hydraulic drive mechanisms, magnetic drivesystems, and fluidic drive systems. The drive mechanism provides a meansfor fluid control within the cartridge and allows for transport of fluidand substances between chambers. The drive mechanism can be used toinitiate and control fluid flow, open valves, form bubbles (e.g. formixing) and to initiate mechanical/chemical processes within thecartridge.

The drive mechanism can also be operably associated with a controller sothat the controller engages the drive mechanism at certain stages in thepathogen capture process. The controller may engage with one or moresensors to determine when and how to activate the drive mechanism duringsample processing. In certain aspects, the controller is a computingsystem. In certain embodiments, drive mechanism is a pneumatic. Thepneumatic drive mechanism can include pumps, electromechanical valves,pressure regulators, tubing, pneumatic manifolds, flow and pressuresensors. Pneumatic drive mechanisms use air pressure and airdisplacement to control the flow of fluids within the cartridge. Incertain aspects, the pneumatic drive mechanism is coupled to electronicregulators. When coupled to an electronic regulator, the pneumaticmechanism may be an external compressor with a reservoir for pumpingcompressed nitrogen, argon or air.

The instrument also includes one or more magnetic assemblies. Themagnetic assemblies engage with one or more magnetic traps (typically,flow-through chambers) of the cartridge. The magnetic assemblies caninclude permanent magnets, removable magnets, electromagnets, or thelike, or combinations thereof. The magnet assemblies may have magnets ofvarious shapes, and of varying strengths, depending on the applicationthereof. If the instrument includes electromagnets, i.e. magnets thatproduce a magnetic field upon introduction of an electric current, theinstrument may also include a current generator to activate theelectromagnets. Depending on the stage of processing, the magneticassembly includes one or magnet that are positioned against thecartridge to facilitate capture of one or more magnetic particles on asurface of a magnetic trap. Alternatively, the electromagnets can beprepositioned next to the cartridge and activated by an electric currentto facilitate capture of one or more magnetic particles against thesurface of the trap.

The size and strength of the magnet(s) of the magnetic assembly shouldproduce a magnetic field suffice to force the magnetic particles withinthe sample against a surface of the magnetic trap of the cartridge,either macrofluidic or mircofluidic. For example, the magnetic assemblycan include 7 bar NdFeB magnets that can be positioned against amagnetic trap of the cartridge. In another example, the magnet assemblyincludes a magnet with a magnetic flux of about 0.6 T and a magneticgradient of about 150 T/m. This magnet's high magnetic gradient of about150 T/m is capable of isolating a plurality of magnetic particles (forexample, 1000 magnetic particles) in on a surface with a micro-scalesurface area.

The instrument can also include mechanical, electrical, andthermo-electrical systems. For instance, instrument can includemechanical mechanism for engaging with a paddle mixer disposed within ina mixing chamber of the cartridge. The instrument can also includepistons and plungers to activate one or more push valves located on thecartridge. In addition, the instrument can include a heating systemdesigned to control the temperatures of one or more components of thecartridge. For example, the instrument can include a heating apparatusoperably associated with the mixing chamber to heat the chamber andencourage binding of one or more magnetic particles with targetscontained within the sample. The instrument may include a controlprocessor or a computing system to activate other subsystems, such asthe drive mechanism. The control processor can be keyed into sensorsdesigned to track the process through the cartridge. This allows thecontrol processor to activate certain substances based on the locationof the fluid within the cartridge or based upon the stage of processing.

The instrument can also include a lysing mechanism for invoking lysis ofcells within the sample. The lysing mechanism can include any sonicationdevice that is well-known in the art. In certain embodiments, thesonication device is the VCX 750 Sonicator sold under the trademarkVIBRA-CELL (sonicator, commercially available from Sonics and Materials,Inc.). Generally, the probe of the sonicator is placed into the liquidcontaining the targets to be lysed. Electrical energy from a powersource is transmitted to a piezoelectric transducer within the sonicatorconverter, where it is changed to mechanical vibrations. Thelongitudinal vibrations from the converter are intensified by the probe,creating pressure waves in the liquid. These in turn produce microscopicbubbles, which expand during the negative pressure excursion and implodeviolently during the positive excursion. This phenomenon, referred to ascavitation, creates millions of shock waves and releases high levels ofenergy into the liquid, thereby lysing the target. In anotherembodiment, the sonication transducer may be brought in contact with achamber holding captured complexes by way of a structural interface. Thesonication transducer vibrates structural interface, such as a thin filmbetween the magnetic trap and the transducer, until lysis is achieved.In either method, the appropriate intensity and period of sonication canbe determined empirically by those skilled in the art.

FIGS. 5 and 6 depict the instrument 200 of the target capture system foruse with the cartridge 100. FIG. 5 depicts the cartridge 100 loaded intothe instrument 200. FIG. 6 depicts the instrument 200 without acartridge 100 loaded. The features of the instrument 200 are discussedin detail above.

FIG. 7 depicts another schematic view of the cartridge 100 of the targetcapture system. FIGS. 8-23 depict the process of target capture withinthe target capture system as the sample is directed into and processedwithin the cartridge. The arrows within the figures indicate the path offluid flow.

As shown in FIG. 8, a vessel 10 coupled to and in fluidic and pneumaticcommunication with the cartridge 100 via the output member 150 and inputmember 140. The vessel 10 is an enclosed collection tube containing asample. In one embodiment, vessel contains 10 to 15 mL of blood. Thecartridge 100 is placed within an instrument and connected to theinstrument's 200 drive mechanism at the instrument interface 45 throughinterface ports 190. Once the vessel 10 is in communication with thecartridge 100, the user can activate the instrument to initiate thetarget capture process. The instrument 200 activates and empties achamber containing magnetic particles 40 into a particle bufferreservoir 20. Each of the magnetic particles is conjugated to a moietyspecific to at least one target. As discussed above, the plurality ofmagnetic particles in the chamber 40 may include different sets ofmagnetic particles in which each set is conjugated to capture moietyspecific to a different target of interest (e.g. for multiplex captureof targets). The chamber containing magnetic particles 40 is typically aglass ampoule. A piston located on the instrument can apply pressure tothe particle chamber 40 causing it to release the particles into thebuffer reservoir 20. Alternatively and as shown in FIG. 2, the chambercontaining the magnetic particles 40 is located between the bufferreservoir 20 and the vessel 10. In this configuration, the drivemechanism forces the buffer towards the particle chamber 40 with enoughpressure to cause the buffer to break a seal on the particle chamber 40and introduce the buffer into the particle chamber 40. In eitherembodiment, the result is having the buffer and particles in the samechamber/reservoir. Typically, the buffer/particle mixture is present ina 2:1 ratio to the initial volume of sample (e.g. buffer/particlemixture is about 20 mL to 30 mL for a sample of 10 mL to 15 mL). Anybuffer suitable for promoting binding of magnetic particles (havingbinding moieties specific to targets) to targets is suitable for use inthe cartridge 100. Once in the same chamber, a bubble can be introducedto ensure the magnetic particles are fully submersed in the buffer.

As shown in FIG. 9, prior to introducing the particle/buffer solutioninto the vessel, the drive mechanism of the instrument forces air intothe vessel through output member 150. The air pressure causes at least aportion of the sample to flow through the input member 140 and into thecartridge 100. Within the cartridge 100, the sample is driven through achannel towards the mixing chamber 70. A bubble sensor prior to theinlet of the mixing chamber 70 and air displacement sensor monitor theamount of fluid flowing from the vessel to the mixing chamber 70. Theinstrument can alert an operator if the desired volume of fluidtransferred is not as suspected. In certain embodiments, the drivemechanism forces the entire sample out of the vessel 10 and into thecartridge 100 prior to introducing the buffer/particle mixture into thevessel 10. Alternatively, the drive mechanism transfers none or aportion of the sample into the cartridge 100 prior to introducing thebuffer/particle mixture into the vessel 10. In the addition, the step ofintroducing the particle/buffer mixture into the vessel 10 can besubstantially concurrent with the step of transferring the sample and/orsample/particle/buffer mixture into the cartridge 100.

In FIG. 10, the buffer/particle mixture is transferred out of theparticle buffer reservoir 20 and into the vessel 10 through outputmember 150 via air pressure from the drive mechanism. Thebuffer/particle mixture is transported into the vessel 10 withsufficient force to hit the top of the vessel 10 so that thebuffer/particle mixture rinses down the sides of the vessel 10. Thisensures any sample collected on the sides of the vessel 10 is introducedinto the cartridge 100 through the input member 10. Thesample/particle/buffer mixture is driven into the cartridge 100 throughinput member 140. In one embodiment, after substantially the entiresample and/or particles have been transferred into the cartridge, bufferis still introduced in to the cartridge to conduct an additional rinse.In certain embodiments, after transfer of the buffer into the vessel anddraining of buffer/sample/particle mixture into the cartridge, the drivemechanism continues to force air into the vessel 10 to ensure anyresidual fluid is moved into cartridge 100. The process depicted in FIG.10 increases the amount of initial sample that is introduced into thecartridge for processing. Because pathogens are often present in levelsas 1 CFU/mL, the ability to transfer all of the initial sample fluidinto the cartridge advantageously prevents the chance that samplecontaining the pathogen would remain in the vessel.

Once the sample/particle/buffer mixture is transferred from the vessel10 to the mixing chamber 70 as shown in FIG. 11, the mixture is agitatedand incubated in the mixing chamber 70. For agitation, the instrument200 rotates the mixer paddle 170. In one embodiment, the instrument 200heats the mixing chamber 70 to a temperature ideal for promoting bindingof the magnetic particles and any targets present within the sample. Theincubation/agitation process is to form target/particle complexes withinthe fluid. A temperature sensor can be included in the mixing chamber 70to monitor temperature. The amount of time the sample/particle/buffermixture is in the mixing chamber 70 can depend on a variety of factors.For example, incubation and agitation time will depend on the desireddegree of binding between the pathogen and the compositions of theinvention (e.g., the amount of moment that would be desirably attachedto the pathogen), the amount of moment per target, the amount of time ofmixing, the type of mixing, the reagents present to promote the bindingand the binding chemistry system that is being employed. Incubation timecan be anywhere from about 5 seconds to a few days. Exemplary incubationtimes range from about 10 seconds to about 2 hours. Binding occurs overa wide range of temperatures, generally between 15° C. and 40° C.

After incubation/agitation, the sample/particle/buffer mixture is cycledthrough the first magnetic trap 60, as shown in FIG. 12. During thecycling process, a magnetic assembly of the instrument engages with thefirst magnetic trap 60 to generate a magnetic field that captures themagnetic particles from the mixture cycled there through on a surface ofthe first magnetic trap 60. In one embodiment, the magnetic trap 60 hasa flow path cross-section of 0.5 mm×20 mm (h×w) and the magneticassembly is an array of bar NdFeB magnets positioned perpendicular tothe flow of fluid into the magnetic trap. For cycling, the drivemechanism uses air pressure to force the sample/particle/buffer mixturefrom the mixing chamber 70 through the first magnetic trap 60 and to thefirst magnetic trap overflow chamber 50. Once the fluid is passedthrough the magnetic trap 60 and into the overflow chamber 50, the fluidis then moved back through the first magnetic trap 60 and into themixing chamber 70. The fluid can be cycled back and forth between themixing chamber 70 and overflow chamber 50 multiple times and atdifferent flow rates to ensure all magnetic particles are captured. Inone embodiment, a bubble sensor 230 associated with the first magnetictrap is used to detect when fluid entering or exiting the magnetic trapis replaced with air. This alerts the instrument 200 that the fluid fromthe mixing chamber 70 has substantially transferred through the firstmagnetic trap 60 and into the magnetic trap overflow chamber 50. Oncealerted, the fluid is moved back through the first magnetic trap 60. Thebubble sensor 230 can be placed at the entrance of the first magnetictrap 60 (as shown in FIG. 12) or at the exit of the first magnetic trap.As an alternative to the bubble sensor, the cycling of fluid can be timecontrolled.

After the final cycle of fluid through the first magnetic trap 60, theremaining fluid (sample/buffer) separated from the captured magneticparticles is moved into the mixing chamber 70 and stored as waste.Alternatively, the remaining fluid can be transferred to a designatedwaste chamber.

The captured particles within the first magnetic trap 60 are thensubject to a wash process as shown in FIG. 13. During the wash process,the magnetic assembly is still engaged with the first magnetic trap 60.A wash solution from wash solution reservoir 35 is transferred into thefirst magnetic trap 60 until the first magnetic trap 60 is filled. Asshown in FIG. 14, the wash solution is then moved out of the firstmagnetic trap 60, thereby washing/rinsing the particles captured on thesurface of the first magnetic trap 60. The wash solution can be movedinto the mixing chamber 70 and stored as waste or into a designatedwaste chamber. Pressure can used to avoid back flow of fluid into thefirst magnetic trap 60.

As shown in FIG. 15, the first magnetic trap 60 is further rinsed tosuspend captured particles in a fluid and to move the magnetic particlesinto the second magnetic trap 80. The drive mechanism moves additionalwash solution into the first magnetic trap 60, in which one opening ofthe magnetic trap is closed to prevent flow through. During introductionof wash solution to the first magnetic trap, the magnetic particles arereleased from the surface and suspended in the wash solution. To removethe magnetic particles from the surface of the first magnetic trap 60,the magnetic assembly is removed from against the first magnetic trap 60or the magnetic assembly is no longer energized. In one embodiment, aswiper magnet engages an opposite side of the first magnetic trap 60 toencourage the particles to re-suspend. The fluid with the particles fromthe first magnetic trap 60 is then transported into the second magnetictrap 80. The second magnetic trap 80 is a flow through chamber. Prior totransfer of particles through the second magnetic trap 80, a magneticassembly engages with the second magnetic trap 80. This magneticassembly can be the same as or different from the magnetic assembly thatengaged with the first magnetic trap 60. In one embodiment, the magneticassembly is different and includes one or more magnets that emit amagnetic field capable of isolating the quantity of magnetic particlestransferred from the first magnetic trap 60 within the second magnetictrap 80 having a microfluidic volume. For example, a second magnetictrap 80 having a volume 500 μL can engage with a magnet having amagnetic flux of 0.6 T and a magnetic gradient of 150 T/m to isolateabout 1000 particles (assuming 1000 particles were initially introducedinto the sample, processed through the first magnetic trap 60, andtransferred to the second magnetic trap 80).

The second magnetic trap 80, as engaged with the magnetic assembly,captures magnetic particles as the fluid flows from the first magnetictrap 60 through the second magnetic trap 80 and into a waste chamber105. Pressure from the drive mechanism is applied to ensure all thefluid/magnetic particles are transferred into the magnetic trap and toprevent any fluid back flow. The rate of the fluid flow can becontrolled to ensure all magnetic particles are capture while the fluidflows through the second magnetic trap 80. In one embodiment, the rateof fluid flow is 1 mL/min. In one aspect, the second magnetic trap 80has a significantly smaller volume than the first magnetic trap 60 whichallows the second magnetic trap 80 to concentrate the substantially theentire quantity of particles initially introduced into the sample into asmall volume of fluid. The high concentration of particles in a smallvolume of fluid provides for easier downstream analysis of or processesperformed on targets bound to those particles. That is, the targetcapture system is able to isolate the clinically relevant portion of amacrofluidic fluid volume in a microfluidic fluid volume. In oneembodiment, the first magnetic trap 80 is macrofluidic (volume capacityabove 1 mL) and the second magnetic trap is microfluidic (volumecapacity below 1000 μL). For example, the first magnetic trap 60 has amacrofluidic volume for processing 30 mL of fluid to initially capturemagnetic particles disposed within the 30 mL of fluid and the secondmagnetic trap 80 has a microfluidic volume of 500 μL of less.

After the magnetic particles are concentrated in the second magnetictrap 80, the captured particles can be directed to a capture vial orsubject to further processing. FIG. 16 shows the transfer of thecaptured particles from the second magnetic trap 80 to the capture vial170. For transfer to the capture vial 170, the magnet assembly isdisengaged from the second magnetic trap 80. Then, a target culturebuffer from a reservoir 280 flushes the contents of the second magnetictrap 80 into the capture vial 170. This provides for direct capture ofthe particles and molecules attached thereto. Direct capture ofparticles from the second magnetic trap 80 can be used to capture wholelive cell targets bound to the particles.

FIGS. 17-22 depict further processing of captured particles to obtainnucleic acids from any targets bound to the captured particles. First,the captured magnetic particles are subject to sonication to invoke celllysis of target cells bound to the particles. This step allows lysis oftarget cells in the presence of the magnetic particles withoutpre-separation of the particles from the target. This step avoidspotential loss of targets during the pre-separation step. As shown inFIG. 17, a fluid from reservoir 90 is transferred into the secondmagnetic trap 80. In one embodiment, the fluid is a lysing buffer oragent. The outlet port of the second magnetic trap 80 is closed to allowthe cell lysis buffer to fill the second magnetic trap 80. Once filled,the magnetic assembly is disengaged from the second magnetic trap 80 tosuspend the magnetic particles in the buffer. A sonicator probe (such asa VibraCell, sonicator commercially available from Sonics and Materials,Inc.) of the instrument is positioned against a surface of the secondmagnetic trap 80. In one embodiment, the surface is a formed from a thinfilm, such as a Mylar film of 125 μM. The sonicator can be activated fordifferent lengths and at different settings to achieve cell lysis. Inone embodiment, the cartridge 100 further includes a reservoir of lysisbashing beads that can be transferred into the second magnetic trap 80to assist with cell lysis.

After lysis by sonication is complete, the lysate can be forced into apre-column mixer 85 as shown in FIG. 18. A nucleic acid extractionbuffer can also be introduced into the pre-column mixer 85 fromreservoir 75. The lysate and the extraction buffer are mixed within thepre-column mixer 85. In one embodiment, the pre-column mixer 85 is abubble mixer and the lysate/extraction buffer mixture is agitated viabubbling air into the mixer 85. FIG. 19 depicts the lysate/extractionbuffer mixture is transferred from the pre-column mixer 85 throughnucleic acid extraction matrix 110 and into a waste chamber 95. In oneembodiment, the nucleic acid extraction matrix 110 is an affinitycolumn. The nucleic acid extraction matrix 110 retains nucleic acidsfrom the lysate/extraction buffer mixture.

The nucleic acid extraction matrix 110 can then be subject to one ormore washes. As shown in FIGS. 20 and 21, the nucleic acid extractionmatrix 95 is subject to two washes. Air pressure forces wash buffersfrom chambers 65 through the nucleic acid extraction member 110 and intowaste chamber 95 to remove any unwanted particulates. In one embodiment,any remaining volatiles within the nucleic acid extraction matrix 110are removed with air pressure from the drive mechanism.

After the washes, the nucleic acid extraction matrix 110 can be elutedwith a fluid from the elution reservoir 55. In one embodiment, the fluidis water. The drive mechanism uses high air pressure to force the fluidthrough the nucleic acid extraction matrix 110 into a nucleic acidcapture vial 180 (SEE FIG. 22). This elution step transfers extractednucleic acids into the vial. The capture vial 180 can be removed fromthe cartridge by the operator and subject to further analysis. Incertain embodiments, the capture vial 180 may include one or morecomponents required for subsequent analysis of the extracted nucleicacids. For example, the one or more components may include primer/probesets for real-time PCR quantification. Preferably, the one or morecomponents for subsequent analysis of the extracted nucleic acids arespecific to same pathogens that the magnetic particles were designed tocapture.

Detection of Target Cell or Detection of Nucleic Acids

In particular embodiments, the isolated targets or the extracted nucleicacid from the captured targets, as isolated with the target capturesystem, may be analyzed by a multitude of technologies. Thesetechnologies include, for example, miniature NMR, Polymerase ChainReaction (PCR), mass spectrometry, fluorescent labeling andvisualization using microscopic observation, fluorescent in situhybridization (FISH), growth-based antibiotic sensitivity tests, andvariety of other methods that may be conducted with purified targetwithout significant contamination from other sample components.

In one embodiment, isolated bacteria are eluted from the magneticparticles and are lysed with a chaotropic solution, and DNA is bound toDNA extraction resin. After washing of the resin, the bacterial DNA iseluted and used in quantitative RT-PCR to detect the presence of aspecific species, and/or, subclasses of bacteria.

In another embodiment, captured bacteria is removed from the magneticparticles to which they are bound and the processed sample is mixed withfluorescent labeled antibodies specific to the bacteria or fluorescentGram stain. After incubation, the reaction mixture is filtered through0.2 μm to 1.0 μm filter to capture labeled bacteria while allowingmajority of free particles and fluorescent labels to pass through thefilter. Bacteria is visualized on the filter using microscopictechniques, e.g. direct microscopic observation, laser scanning or otherautomated methods of image capture. The presence of bacteria is detectedthrough image analysis. After the positive detection by visualtechniques, the bacteria can be further characterized using PCR orgenomic methods.

Detection of bacteria of interest can be performed by use of nucleicacid probes following procedures which are known in the art. Suitableprocedures for detection of bacteria using nucleic acid probes aredescribed, for example, in Stackebrandt et al. (U.S. Pat. No.5,089,386), King et al. (WO 90/08841), Foster et al. (WO 92/15883), andCossart et al. (WO 89/06699), each of which is hereby incorporated byreference.

A suitable nucleic acid probe assay generally includes sample treatmentand lysis, hybridization with selected probe(s), hybrid capture, anddetection. Lysis of the bacteria is necessary to release the nucleicacid for the probes. The nucleic acid target molecules are released bytreatment with any of a number of lysis agents, including alkali (suchas NaOH), guanidine salts (such as guanidine thiocyanate), enzymes (suchas lysozyme, mutanolysin and proteinase K), and detergents. Lysis of thebacteria, therefore, releases both DNA and RNA, particularly ribosomalRNA and chromosomal DNA both of which can be utilized as the targetmolecules with appropriate selection of a suitable probe. Use of rRNA asthe target molecule(s), may be advantageous because rRNAs constitute asignificant component of cellular mass, thereby providing an abundanceof target molecules. The use of rRNA probes also enhances specificityfor the bacteria of interest, that is, positive detection withoutundesirable cross-reactivity which can lead to false positives or falsedetection.

Hybridization includes addition of the specific nucleic acid probes. Ingeneral, hybridization is the procedure by which two partially orcompletely complementary nucleic acids are combined, under definedreaction conditions, in an anti-parallel fashion to form specific andstable hydrogen bonds. The selection or stringency of thehybridization/reaction conditions is defined by the length and basecomposition of the probe/target duplex, as well as by the level andgeometry of mis-pairing between the two nucleic acid strands. Stringencyis also governed by such reaction parameters as temperature, types andconcentrations of denaturing agents present and the type andconcentration of ionic species present in the hybridization solution.

The hybridization phase of the nucleic acid probe assay is performedwith a single selected probe or with a combination of two, three or moreprobes. Probes are selected having sequences which are homologous tounique nucleic acid sequences of the target organism. In general, afirst capture probe is utilized to capture formed hybrid molecules. Thehybrid molecule is then detected by use of antibody reaction or by useof a second detector probe which may be labelled with a radioisotope(such as phosphorus-32) or a fluorescent label (such as fluorescein) orchemiluminescent label.

Detection of pathogen of interest can also be performed by use of PCRtechniques. A suitable PCR technique is described, for example, inVerhoef et al. (WO 92/08805). Such protocols may be applied directly tothe pathogen captured on the magnetic particles. The pathogen iscombined with a lysis buffer and collected nucleic acid target moleculesare then utilized as the template for the PCR reaction.

In certain embodiments, nucleic acids derived from the captured pathogenare analyzed in a multiplex reaction in order to rapidly detect two ormore pathogens present in the sample. Any multiplex reaction known inthe art may be used, such as multiplex ELISA, multiplex sequencing,multiplex probe hybridization, etc. In certain embodiments, multiplexreactions may involve the amplification and quantification of two ormore targets in the same reaction volume. Typical multiplex reactionsinvolve PCR, qPCR (real-time PCR), and sequencing.

Multiplex PCR refers to the use of more than one primer pair in a singlereaction vessel in order to amplify more than one target sequence. Inthe case of real-time PCR, more than one primer pair/probe set isutilized. Multiplex PCR allows for simultaneous detection of differenttargets in the same reaction. The target sequences may be identified byan identifiable label (e.g. fluorescent probe) or by subsequentsequencing. Multiplex real-time PCR uses multiple probe-based assays, inwhich each assay has a specific probe labeled with a unique fluorescentdye, resulting in different observed colors for each assay. Real-timePCR instruments can discriminate between the fluorescence generated fromdifferent dyes. Different probes are labeled with different dyes thateach have unique emission spectra. Spectral signals are collected withdiscrete optics, passed through a series of filter sets, and collectedby an array of detectors. Spectral overlap between dyes is corrected byusing pure dye spectra to deconvolute the experimental data by matrixalgebra. An overview of real-time PCR techniques is described in detailin Elnifro, Elfath M., et al. “Multiplex PCR: optimization andapplication in diagnostic virology.” Clinical Microbiology Reviews 13.4(2000): 559-570. Multiplex PCR reaction techniques are described in U.S.Publication Nos. 20130059762, and 2005/0026144 as well as Carroll, N.M., E. E. Jaeger, et al. (2000). “Detection of and discriminationbetween gram-positive and gram-negative bacteria in intraocular samplesby using nested PCR.” J Clin Microbiol 38(5): 1753-1757, and Klaschik,S., L. E. Lehmann, et al. (2002). “Real-time PCR for detection anddifferentiation of gram-positive and gram-negative bacteria.” J ClinMicrobiol 40(11): 4304-4307.

Multiplex sequencing involves the simultaneous sequencing of multipletarget sequences in a single sequencing run. Multiplex sequencing allowsfor differentiation between target sequences of different pathogens in asample and differentiation between nucleic acid sequences of pooledsamples (e.g. differentiate between two or more patient samples). Inorder to differentiate between target sequences, one or more differentbarcodes may be introduced to the nucleic acid of a sample. Earlymultiplex sequencing is described in more detail in G. M. Church in U.S.Pat. No. 4,942,124 and further by G. M. Church and S. Kieffer-Higgins inU.S. Pat. No. 5,149,625. Multiplex sequencing of multiple samplesinvolves sequencing of a plurality of template nucleic acid moleculesfrom different samples at the same time on the same platform byattaching a unique oligonucleotide sequence (i.e., a bar code) to thetemplate nucleic acid molecules from different samples prior to poolingand sequencing of the template molecules. The bar code allows fortemplate nucleic acid sequences from different samples to bedifferentiated from each other. Once bar coded, template molecules fromdifferent samples may be pooled and sequenced at the same time on thesame platform. Because the bar code on each template molecule issequenced as part of the sequencing reaction, the bar code is acomponent of the sequence data, and thus the sequence data for differentsamples is always associated with the sample from which it originated.Due to the association in the sequence data, the sequence data from thepooled samples may be separated after sequencing has occurred andcorrelated back to the sample from which it originated.

Any sequencing technique (for singleplex and multiplex assays) may beutilized to identify isolated pathogens by their nucleic acid extracts.Suitable sequencing techniques include, for example, classic dideoxysequencing reactions (Sanger method) using labeled terminators orprimers and gel separation in slab or capillary, sequencing by synthesisusing reversibly terminated labeled nucleotides, pyrosequencing, 454sequencing, allele specific hybridization to a library of labeledoligonucleotide probes, sequencing by synthesis using allele specifichybridization to a library of labeled clones that is followed byligation, real time monitoring of the incorporation of labelednucleotides during a polymerization step, polony sequencing, and SOLiDsequencing. Sequencing of separated molecules has more recently beendemonstrated by sequential or single extension reactions usingpolymerases or ligases as well as by single or sequential differentialhybridizations with libraries of probes.

For detection of the selected pathogen by use of antibodies, isolatedpathogen are contacted with antibodies specific to the pathogen ofinterest. As noted above, either polyclonal or monoclonal antibodies canbe utilized, but in either case have affinity for the particularpathogen to be detected. These antibodies, will adhere/bind to materialfrom the specific target pathogen. With respect to labeling of theantibodies, these are labeled either directly or indirectly with labelsused in other known immunoassays. Direct labels may include fluorescent,chemiluminescent, bioluminescent, radioactive, metallic, biotin orenzymatic molecules. Methods of combining these labels to antibodies orother macromolecules are well known to those in the art. Examplesinclude the methods of Hijmans, W. et al. (1969), Clin. Exp. Immunol. 4,457-, for fluorescein isothiocyanate, the method of Goding, J. W.(1976), J. Immunol. Meth. 13, 215-, for tetramethylrhodamineisothiocyanate, and the method of Ingrall, E. (1980), Meth. in Enzymol.70, 419-439 for enzymes.

These detector antibodies may also be labeled indirectly. In this casethe actual detection molecule is attached to a secondary antibody orother molecule with binding affinity for the anti-bacteria cell surfaceantibody. If a secondary antibody is used it is preferably a generalantibody to a class of antibody (IgG and IgM) from the animal speciesused to raise the anti-bacteria cell surface antibodies. For example,the second antibody may be conjugated to an enzyme, either alkalinephosphatase or to peroxidase. To detect the label, after the bacteria ofinterest is contacted with the second antibody and washed, the isolatedcomponent of the sample is immersed in a solution containing achromogenic substrate for either alkaline phosphatase or peroxidase. Achromogenic substrate is a compound that can be cleaved by an enzyme toresult in the production of some type of detectable signal which onlyappears when the substrate is cleaved from the base molecule. Thechromogenic substrate is colorless, until it reacts with the enzyme, atwhich time an intensely colored product is made. Thus, material from thebacteria colonies adhered to the membrane sheet will become an intenseblue/purple/black color, or brown/red while material from other colonieswill remain colorless. Examples of detection molecules includefluorescent substances, such as 4-methylumbelliferyl phosphate, andchromogenic substances, such as 4-nitrophenylphosphate,3,3′,5,5′-tetramethylbenzidine and2,2′-azino-di-[3-ethelbenz-thiazoliane sulfonate (6)]. In addition toalkaline phosphatase and peroxidase, other useful enzymes includeβ-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase,α-mannosidase, galactose oxidase, glucose oxidase and hexokinase.

Detection of bacteria of interest using NMR may be accomplished asfollows. In the use of NMR as a detection methodology, in which a sampleis delivered to a detector coil centered in a magnet, the target ofinterest, such as a magnetically labeled bacterium, may be delivered bya fluid medium, such as a fluid substantially composed of water. In sucha case, the magnetically labeled target may go from a region of very lowmagnetic field to a region of high magnetic field, for example, a fieldproduced by an about 1 to about 2 Tesla magnet. In this manner, thesample may traverse a magnetic gradient, on the way into the magnet andon the way out of the magnet. As may be seen via equations 1 and 2below, the target may experience a force pulling into the magnet in thedirection of sample flow on the way into the magnet, and a force intothe magnet in the opposite direction of flow on the way out of themagnet. The target may experience a retaining force trapping the targetin the magnet if flow is not sufficient to overcome the gradient force.m dot (del B)=F  Equation 1v _(t) =−F/(6*p*n*r)  Equation 2where n is the viscosity, r is the particle diameter, F is the vectorforce, B is the vector field, and m is the vector moment of the particle

The detection method is based on a miniature NMR detector tuned to themagnetic resonance of water. When the sample is magnetically homogenous(no bound targets), the NMR signal from water is clearly detectable andstrong. The presence of magnetic material in the detector coil disturbsthe magnetic field, resulting in reduction in water signal. One of theprimary benefits of this detection method is that there is no magneticbackground in biological samples which significantly reduces therequirements for stringency of sample processing. In addition, since thedetected signal is generated by water, there is a built-in signalamplification which allows for the detection of a single labeledbacterium. NMR detection is described in further detail in co-pendingand co-assigned U.S. application Ser. No. 13/091,506.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

What is claimed is:
 1. A method for isolating a pathogen from a sample,the method comprising removably coupling a vessel comprising apathogen-containing sample to a cartridge via a cartridge interface thatis configured to provide communication between the vessel and thecartridge; flowing magnetic particles conjugated to a capture moietyfrom the cartridge into the vessel to form a mixture; flowing themixture from the vessel into the cartridge; flowing a fluid from thecartridge into the vessel to flush residual mixture from the vessel intothe cartridge; and incubating the mixture in the cartridge to allow theparticles to bind to the pathogen; applying a magnetic field to capturepathogen/magnetic particle complexes on a surface of a first magnetictrap; washing the captured pathogen/magnetic particle complexes with awash solution, thereby isolating the pathogen/magnetic particlecomplexes; removing the magnetic field; re-introducing the washsolution, thereby re-suspending the pathogen/magnetic particlecomplexes; flowing the re-suspended pathogen/magnetic particle complexesthrough a channel and into a second magnetic trap; and engaging thesecond magnetic trap, thereby re-capturing the pathogen/magneticparticle complexes; wherein the first magnetic trap is configured tocapture the pathogen/magnetic particle complexes from a macro-scalevolume of fluid, and the second magnetic trap is configured toconcentrate substantially all of the pathogen/magnetic particlecomplexes into a micro-scale volume of fluid.
 2. The method of claim 1,further comprising lysing the captured pathogen in a region selectedfrom the group consisting of the first magnetic trap, the secondmagnetic trap, and a combination thereof.
 3. The method of claim 2,further comprising separating nucleic acid from the lysate; andcollecting the separated nucleic acid into a vial.
 4. The method ofclaim 3, further comprising analyzing the nucleic acid to therebyidentify the pathogen.
 5. The method of claim 4, wherein analyzingcomprises conducting a sequencing reaction.
 6. The method of claim 4,wherein analyzing comprises conducting an amplification reaction.
 7. Themethod of claim 6, wherein the vial comprises at least one detectablylabeled nucleic acid probe specific for the pathogen.
 8. The method ofclaim 1, wherein the sample comprises a plurality of pathogen.
 9. Themethod of claim 8, wherein the plurality of magnetic particles arecomposed of different sets, wherein members of the different sets areconjugated to different capture moieties that are specific for thedifferent pathogen.
 10. The method of claim 9, further comprisingconducting a multiplex reaction to analyze nucleic acid from theplurality of isolated pathogen and thereby identify the plurality ofpathogen.
 11. The method of claim 10, wherein the multiplex reaction isa multiplex sequencing reaction.
 12. The method of claim 10, wherein themultiplex reaction is a multiplex amplification reaction.
 13. The methodof claim 1, wherein the capture moiety is a type selected from the groupconsisting of antibodies, lectins, bacteriophages, antimicrobial agents,oligonucleotides, and combinations thereof.
 14. The method of claim 1,wherein the pathogen is selected from the group consisting of fungi,bacteria or both.
 15. The method of claim 14, wherein the fungi areselected from the group consisting of the Candida genus, the Aspergillusgenus, the Cryptococcus genus, and a combination thereof.
 16. The methodof claim 14, wherein the bacteria are selected from the group consistingof the Staphylococcus genus, the Enterobacteriaceae genus, theAcinetobacter genus, the Stenotrophomas genus, the Pseudomonas genus,the Neisseria genus, the Clostridium genus, and the Enterococcus genus.17. The method of claim 1, wherein said sample is a human tissue or bodyfluid.